Feed supplement with lipase and phytase

ABSTRACT

The present invention relates to a feed supplement comprising a phytase and a lipolytic enzyme, wherein said lipolytic enzyme has lipase activity at a pH in the range of about pH1.5 to about pH3.5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S. application Ser. No. 13/265,725, filed Oct. 21, 2011, entitled “Feed Supplement,” now U.S. Pat. No. 8,445,250 which claims priority to PCT Application No. PCT/IB2010/051804 entitled “Feed Supplement,” filed Apr. 23, 2010, which claims priority to U.S. Provisional Application No. 61/172,272, filed Apr. 24, 2009, Great Britain Application No. 0908770.1, filed May 20, 2009, Great Britain Application No. 0922467.6, filed Dec. 23, 2009, and U.S. Provisional Application No. 61/312,413, filed Mar. 10, 2010, all of which are expressly incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to feed supplements. More specifically, the present invention relates to feed supplements comprising phytases and lipolytic enzymes which can be used for enhancing digestion in animal feedstuffs and to feedstuffs comprising the feed supplements.

SEQUENCE LISTING

A text file in compliance with ASCII and having a “.txt” extension has been electronically submitted via EFS-Web. The text file named “Sequence Listing—Feed Supplement” was created on Oct. 20, 2011, and is 95,302 bytes. The text file is expressly incorporated by reference herein in its entirety.

BACKGROUND

Phytate is the major storage form of phosphorus in cereals and legumes. However, monogastric animals such as pigs, poultry and fish are not able to metabolise or absorb phytate (or phytic acid) and therefore it is excreted, leading to phosphorous pollution in areas of intense livestock production. Moreover, phytic acid also acts as an antinutritional agent in monogastric animals by chelating metal agents such as calcium, copper and zinc.

In order to provide sufficient phosphates for growth and health of these animals, inorganic phosphate is added to their diets. Such addition can be costly and further increases pollution problems.

Through the action of phytase, phytate is generally hydrolysed to give lower inositol-phosphates and inorganic phosphate. Phytases are useful as additives to animal feeds where they improve the availability of organic phosphorus to the animal and decrease phosphate pollution of the environment (Wodzinski R J, Ullah A H. Adv Appl Microbiol. 42, 263-302 (1996)).

The addition of phytase to broiler feed has also been shown to increase the apparent metabolisable energy (AME), the availability of nitrogen and amino acids (Ravindran, V. et al, Brit. Poultry Sci. 41, 193-200 (2000)).

A number of phytases of fungal (Wyss M. et al. Appl. Environ. Microbiol. 65 (2), 367-373 (1999); Berka R. M. et al. Appl. Environ. Microbiol. 64 (11), 4423-4427 (1998); Lassen S. et al. Appl. Environ. Microbiol. 67 (10), 4701-4707 (2001)) and bacterial (Greiner R. et al Arch. Biochem. Biophys. 303 (1), 107-113 (1993); Kerovuo et al. Appl. Environ. Microbiol. 64 (6), 2079-2085 (1998); Kim H. W. et al. Biotechnol. Lett. 25, 1231-1234 (2003); Greiner R. et al. Arch. Biochem. Biophys. 341 (2), 201-206 (1997); Yoon S. J. et al. Enzyme and microbial technol. 18, 449-454 (1996); Zinin N. V. et al. FEMS Microbiol. Lett. 236, 283-290 (2004)) origin have been described in the literature.

However, fungal phytases tend to be proteolytically unstable (Igbasan F. A. et al. Arch. Anim. Nutr. 53, 353-373 (2000)) and therefore susceptible to degradation, while some bacterial phytases have a narrow substrate specificity for phytate alone and poorly degrade inositol phosphates of intermediate degrees of phosphorylation (Greiner R. et al., Arch. Biochem. Biophys. 303 (1), 107-113 (1993); Kerovuo J et al, Biochem. J. 352, 623-628 (2000)).

Furthermore, it is known that the interaction of calcium with phytate to form Ca-phytate complexes is detrimental to phytase activity (Selle, P. H. et al, Livestock Science. 124, 126-141 (2009)). The calcium in these Ca-phytate complexes has been hypothesised to make phytate inaccessible to the phytase or to compete with non-complexed phytate for the active site of the enzyme (Long, C., Phytase, Biochemists Handbook, Princeton, N.Y., Van Nostrand-Reinhold (1961); Wise, A., Nutrition Abstracts & Reviews, 53, 791-806 (1983)).

The addition of phytase has been shown to increase the AME of a high phytate diet. Ravindran et al (Br. Poult. Sci., 2000, 41, 193-200) has suggested that calcium phytate complexes react with fatty acids to form insoluble soaps in the gut lumen, thereby lowering fat digestibility. The addition of phytase has been suggested to reduce the level of these soaps. Supporting this hypothesis, there is evidence of phytate interactions with lipid in corn (Cosgrove, 1966, Rev. Pure App. Chem. 16:209-224). These ‘lipophytins’ have been described as a complex of Ca/Mg-phytate, lipids and peptides (Cosgrove, 1966, Rev. Pure App. Chem. 16:209-224). Other reports have also suggested interactions between Ca, fat and phytate in the diet. For instance, Matyka et al. (1990) (Anim. Feed. Sci. Technol. 31:223-230) found that dietary tallow reduced phytate P utilization in young chicks, and increased the percentage of fat excreted as soap fatty acids.

The addition of exogenous lipases has had limited success in increasing lipid and mineral digestibility. However, this may have been limited by the formation of complexes of free fatty acids bound with Ca and phytate (Cosgrove, 1966 supra), which are insoluble in the gastro intestinal tract, and poorly absorbed (Matyka et al. 1990 supra).

The use of exogenous lipases in animal feed has been previously suggested with the objective to improve the fat digestion by animals. Lipase is hypothesised to improve digestion as it liberates absorbable free fatty acids (FFA) faster than otherwise would have happened. However, attempts to demonstrate improvements in animal performance or digestibility by the use of exogenous lipases have been at best inconsistent and have often showed lipases not to work. Dierick and Decuypere (2004) (J. Sci. Food Agric. 84:1443-1450) showed no improvements in fat digestibility with addition of a microbial lipase in pig diets. Hurtado et al. (2000) (Rev. Bras. Zootec. 29:794-802) failed to detect increments of body weight gain, feed efficiency and energy digestibility due to the use of an exogenous lipase in piglets. Additionally, they reported no additive effects in any of these parameters when such lipase was combined with an amylase and a protease. Officer (1995) (Anim. Feed Sci. Technol. 56:55-65) reported no significant changes in body weight gain of feed intake of piglets by the use of two combinations of exogenous enzymes which they described as lipase, proteinase, B-glucanase, amylase and cellulose, and lipase, B-glucanase, hemi-cellulase, pentosanase, cellulose, amylase and proteinase.

In broiler chickens, Meng et al. (2004) (Poult. Sci. 83:1718-1727), when using a bacterial lipase in wheat-based diets, failed to detect any effect of the lipase on apparent digestibility of fat, starch, nitrogen and NSP, as well as AME. Additionally, they rejected the hypothesis that a combination of carbohydrases, including xylanase, glucanase and cellulase, on top of the lipase would increase the lipase effects on nutrient digestibility by reducing viscosity of the digesta and increasing fat digestion and absorption.

Al-Marzooqi and Leeson (1999) tested lipases from animal origin and pancreatic extracts. Although they were able to demonstrate improvements in fat digestibility and reductions on the level of soaps at the faecal levels when these additives were used in the diet, they also reported an anorexic effect (reduction of feed intake), which they explained by possible contaminations with cholecystokinin in this type of extracts. This fact limits its utilization in animal feed and possible improvements in growth or feed efficiency in animals.

One aim of the present invention is to provide a feed supplement which provides improved availability of at least one nutrient or an improvement in the apparent metabolisable energy from a feed material.

SUMMARY

The present invention is based on the surprising discovery that the addition of a feed supplement comprising at least one specific lipolytic enzyme and at least one specific phytase to a feedstuff results in improved uptake of at least one nutrient and or mineral compared to the use of the enzymes individually.

According to the broadest aspect of the present invention there is provided a feed supplement comprising:—

i. at least one lipolytic enzyme; and

ii. at least one phytase.

The inventors have surprisingly discovered that contrary to previous reports (Mulyantini, N. G. A. et al Aust. Poult. Sci. Symp, 17, 305-307 (2005)), the addition of a feed supplement comprising at least one specific phytase and at least one specific lipolytic enzyme to feed material to produce a feedstuff results in an increase in the availability of at least one nutrient and/or an increase in the apparent metabolisable energy (AME).

Although not wishing to be bound by theory, the inventors have hypothesised that this surprising discovery may be due to the action of the lipolytic enzyme liberating free fatty acids (FFA) at an earlier stage in digestion where the pH is acidic. These FFA may bind with excess calcium to form soaps in the gut. There is then less free calcium available to bind to phytate, thereby boosting phytase activity in the regions of the gastrointestinal tract (GIT) where phytic acid and calcium tends to form a phytase resistant complex. This leads to increased phosphate liberation and absorption. The free fatty acid calcium complexes dissolve later in the GIT resulting in increased fat and calcium absorption and/or increased AME.

According to another aspect of the present invention there is provided a method of making a feed supplement comprising mixing at least one lipolytic enzyme and at least one phytase.

According to another aspect of the present invention there is provided a feedstuff comprising a feed material and the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention.

According to a further aspect of the present invention, there is provided a method of making a feedstuff comprising adding to a feed material a feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention.

According to a further aspect of the present invention there is provided a method for increasing the availability of at least one dietary nutrient and/or increasing the apparent metabolisable energy (AME) from a feed material comprising adding to feed material a feed supplement comprising a combination of at least one lipolytic enzyme and at least one phytase or adding to the feed material at least one lipolytic enzyme and at least one phytase.

According to a further aspect of the present invention there is provided a method of increasing the growth rate of an animal comprising feeding the animal an effective amount of a feedstuff according to the present invention.

According to a further aspect of the present invention there is provided the use of at least one phytase and at least one lipase in the manufacture of a feedstuff for increasing the availability of at least one nutrient and/or increasing the available metabolic energy from a feed material.

It will be understood that any of the preferred features disclosed herein is considered to be equally applicable to any of the aspects described above unless explicitly stated otherwise. Any preferred feature is also considered to be disclosed in combination with any other preferred feature disclosed herein.

To be efficient as an enzyme additive to animal feed, a phytase has to combine a number of different properties. In order to be able to degrade phytic acid in the acidic environment of an animal's stomach it has to be active at low pH, preferably over a broad range of pH values. In addition, it has to have high specific activity and preferably high thermostability to enable the protein to withstand high temperatures commonly used in preparation of feedstuffs such as feed pellets.

It is also important that the enzyme has broad substrate specificity allowing it to hydrolyse not only phytate but also intermediate products of phytate degradation such as inositol pentaphosphates, tetraphosphates and triphosphates. Studies on phytate degradation in pigs show that these inositol oligophosphates otherwise remain largely insoluble in the small and large intestine and thus inaccessible to alkaline phosphatases produced by the animal and gut microflora (Schlemmer U. et al. Arch. Anim. Nutr. 55, 255-280 (2001)). Variations in substrate specificity profiles of different enzymes have been identified. For example, inositol-triphosphates generated by the phytase from B. subtilis are essentially resistant to further hydrolysis by this enzyme [Kerovuo J. et al. Biochem J. 352, 623-628 (2000)].

In preferred embodiments, the phytase is preferably E. coli phytase marketed under the name Phyzyme XP™ by Danisco A/S. Alternatively the phytase may be a Buttiauxella phytase, for example, the phytase enzymes taught in WO 2006/043178, WO 2008/097619, WO2008/092901 and PCT/US2009/41011 all of which are incorporated herein by reference.

In a most preferred embodiment, the phytase comprises an E. coli phytase and/or a Buttiauxella sp. phytase. More preferably, the phytase comprises a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or SEQ ID NO: 13; or a polypeptide comprising one or several amino acid additions, deletions and/or substitutions compared to any one of SEQ ID NOs: 1-6 or SEQ ID NO: 13; or a polypeptide having at least 70%, 80%, 90%, 95%, 98% or 99% identity to any one of SEQ ID NOs: 1-6 or SEQ ID NO: 13; or a polypeptide produced by expression of a nucleotide sequence comprising the sequence of SEQ ID NO: 11; or nucleotides 253 to 1483 of SEQ ID NO:14 or a sequence which differs from SEQ ID NO: 11 or nucleotides 253 to 1483 of SEQ ID NO:14 due to the degeneracy of the genetic code; or a sequence comprising one or several nucleotide additions, deletions and/or substitutions compared to the sequence of SEQ ID NO: 11; or nucleotides 253 to 1483 of SEQ ID NO:14; or a sequence which has at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 11 or nucleotides 253 to 1483 of SEQ ID NO:14.

In one preferred embodiment, the phytase has phytase activity at least at a pH in the range of about pH 2.5 to about pH 5.5. More preferably, the phytase has phytase activity at least at a pH in the range of about pH 2.5 to about pH 3.5 and also in the range of about pH 5 to about pH 5.5 when measured by the phytase assay disclosed in the materials and methods section below.

Preferably, the phytase has a specific activity level of at least 100 FTU/mg of enzyme. More preferably, at least, 200, 300, 400, 500, 700, 1000 FTU/mg.

It will be understood that as used herein 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined herein in the phytase assay at pH 5.5 described in the materials and methods section.

In preferred embodiments, the phytase for use in the present invention has a pH optimum in the range of pH 2 to 5.5, preferably 4.0-4.5, retaining at least 50% of the maximum activity over the pH range 2.0-5.5 and/or having a specific activity over 300 FTU/mg.

Preferably, the lipolytic enzyme for use in the present invention has lipase activity at a pH in the range of about pH 1.5 to about pH 5.5 when measured by the lipase assay described in the materials and methods section below. More preferably, the lipolytic enzyme has lipase activity at a pH in the range of about pH 1.5 to about pH 3.5, or about pH 2.0 to about pH 3.5.

It will be understood that the lipolytic enzyme for use in the present invention may be any suitable lipolytic enzyme which, using the assay defined herein, shows lipolytic enzyme activity. Preferably, the lipolytic enzyme has an activity level of at least 100 LIPU/mg of enzyme. More preferably, at least, 200, 300, 400, 500, 700, 1000 LIPU/mg.

As used herein, 1 LIPU (lipase unit) is defined as the amount of enzyme which releases 1 μmol of H⁺ per minute under the conditions described in the lipase assay described in the materials and methods section herein below.

In one preferred embodiment, the lipolytic enzyme for use in the present invention is derived from the filamentous fungus Aspergillus tubingensis as described in WO 98/45453.

Preferably, the lipolytic enzyme comprises; a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 7 or 8; or a polypeptide comprising one or several amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 7 or 8; or a polypeptide having at least 70%, 80%, 90%, 95%, 98% or 99% identity thereto; or a polypeptide which is produced by expression of a nucleotide sequence comprising the sequence of SEQ ID NO: 9 or 10; or a sequence which differs from SEQ ID NO:9 or 10 due to the degeneracy of the genetic code; or a sequence comprising one or several nucleotide additions, deletions and/or substitutions compared to the sequence of SEQ ID NO: 9 or 10 or a sequence which has at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 9 or 10.

Alternatively, the lipase may be selected from the group consisting of Acidic lipase from Pseudomonas gessardii (Ramani, K. et al, J Ind Microbiol Biotechnol (2010) 37:531-535), Acidic lipase from Rhizopus arrhizus (Kumar, K K. et al, Hindustan Antibiot Bull. 1993 February-May; 35(1-2):33-42) (SEQ ID NO: 32, 33), Acidic lipase from Penicillium simplicissimum (Gutarra M. L. E. et al, Bioresource Technology 100 (2009) 5249-5254), Acidic lipase from Aspergillus niger NCIM 1207 (Mhetras N. C. et al, Bioresource Technology 100 (2009) 1486-1490, Pel, H. J., et al, Nat. Biotechnol. 25 (2), 221-231 (2007)) (SEQ ID NO: 27), Mammalian gastric lipases from, for example, human (SEQ ID NO: 17, 18), bovine (SEQ ID NO: 19, 20), mouse (SEQ ID NO: 21, 22), rat (SEQ ID NO: 23, 24) and canines (SEQ ID NO: 25, 26) (Chahinian, H. et al, Biochemistry 2006, 45, 993-1001), Acid lipase from Castor beans (Eastmond, P. J. The Journal of Biological Chemistry, 279 (2004); 45540-45545) (SEQ ID NO: 28, 29), and LIP2 lipase from Yarrowia lipolytica (Aloulou, A. et al, Biochimica et Biophysica Acta 1771 (2007) 228-237) (SEQ ID NO: 30, 31).

As indicated above, the inventors have discovered that the improvement in performance of the feed supplement of the present invention is due to the action of the lipolytic enzyme liberating free fatty acids (FFA) at an earlier stage in digestion. This results in the formation of calcium soaps and the associated reduction in calcium-phytate complexes.

In a further preferred embodiment, the lipolytic enzyme for use in the present invention is produced in a Trichoderma reesei cell, for example by expression of a nucleotide having the sequence of SEQ ID NO: 9 or SEQ ID NO:10 in a T. reesei host cell.

In a further preferred embodiment, the feed supplement according to the present invention comprises:

i) a lipolytic enzyme which is a lipolytic enzyme characterised by at least one of the following characteristics:

-   -   a) a lipolytic enzyme that has lipase activity at a pH in the         range of about pH 1.5 to about pH 3.5 when measured by the         lipase assay disclosed herein;     -   b) a lipolytic enzyme which comprises a polypeptide comprising         the amino acid sequence as shown in SEQ ID NO: 7 or 8 or a         polypeptide having at least 70%, 80%, 90%, 95%, 98% or 99%         identity thereto;     -   c) a lipolytic enzyme which is produced by expression of a         nucleotide sequence comprising the sequence of SEQ ID NO: 9 or         10; or a sequence which differs from SEQ ID NO:9 or 10 due to         the degeneracy of the genetic code; or a sequence which has at         least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 9 or         10; and         ii) a phytase which characterised by at least one of the         following characteristics:—     -   d) a bacterial phytase;     -   e) a phytase that has phytase activity when measured by the         phytase assay described herein below at a pH in the range of         about pH 2.5 to about pH 3.5;     -   f) a phytase which comprises a polypeptide comprising the amino         acid sequence as shown in any one of SEQ ID NOs: 1-6 or SEQ ID         NO:13 or a polypeptide having at least 70%, 80%, 90%, 95%, 98%         or 99% identity thereto;     -   g) a phytase which is produced by expression of a nucleotide         sequence comprising the sequence of SEQ ID NO: 11 or nucleotides         253 to 1483 of SEQ ID NO:14; or a sequence which differs from         SEQ ID NO: 11 or nucleotides 253 to 1483 of SEQ ID NO:14 due to         the degeneracy of the genetic code; or a sequence which has at         least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 11         or nucleotides 253 to 1483 of SEQ ID NO:14.

In a further preferred embodiment, the feed supplement comprises lipolytic enzyme and phytase having a ratio of lipase activity (measured as lipase units (LIPU)) to phytase activity (measured as phytase units (FTU)) in the range of about 1:5 to about 12:1, preferably, in a ratio of about 1:3 to about 3:1 and more preferably in a ratio of about 1:1 to about 3:1 as defined by the lipase and phytase assays at pH5.5 as described herein below.

Preferably, the at least one dietary nutrient whose availability is increased is selected from the group comprising; phosphorous; calcium; amino acids, fat and/or starch. Preferably, the availability is improved both at the ileal and/or the total gastrointestinal tract.

It will be understood by the skilled person that the lipolytic enzyme and the phytase for use in the present invention can be provided independently as either liquid or as solid/granulated compositions.

Preferably, when said enzyme is in liquid form, said enzyme is in the medium into which the enzyme has been secreted following culturing of a cell comprising said enzyme. Preferably said medium is cell-free (i.e. the cell(s) have been separated from the medium). Preferably said medium is concentrated. It will be understood that the medium can be granulated to provide a solid enzyme composition.

It will be further understood that the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention may be provided in the form of a solution or as a solid—depending on the use and/or the mode of application and/or the mode of administration.

In one embodiment the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention is in a liquid formulation suitable for consumption, preferably such liquid composition contains either buffer, salts, sorbitol and/or glycerol.

In an alternative embodiment, feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention can be provided as one or more cells comprising said lipolytic enzyme and/or phytase.

In one embodiment the feed supplement is granulated or co-granulated with other enzymes.

Preferably, the feed supplement further comprises at least one physiologically acceptable carrier.

The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, anti-foam, Na₂SO₄, Talc, PVA and mixtures thereof.

In one embodiment the phytase and/or lipolytic enzyme is dried on the physiologically acceptable carrier.

In preferred embodiments, the feed supplement may comprise at least one further enzyme. In preferred embodiments, the at least one further feed enzyme is selected from the group consisting of those involved in starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, -glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, -glucosidases, including β glucosidase, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: O₂-oxidoreductase, EC 1.1.3.5) β-glucanase, α-amylase, pectinase, cellobiohydrolase, acid phosphatases and/or others or combinations thereof. These include enzymes that, for example, modulate the viscosity of the feed.

In a more preferred embodiment the feed supplement comprises a phytase, a lipolytic enzyme, a xylanse and/or an amylase. In a most preferred embodiment, the feed supplement comprises phytase, a lipolytic enzyme, a xylanse and an amylase.

Preferably, the amylase is present in the range of 10 U/kg to 10000 U/kg feed, more preferably, 100 U/kg to 7500 U/kg, and even more preferably, 500 U/kg to 5000 U/kg. It will be understood that one amylase U is the amount of enzyme that releases 1 mmol of glucosidic linkages from a water insoluble cross-linked starch polymer substrate per min at pH 6.5 and 37° C.

Preferably, the xylanse is present in the range of 100 U/kg to 10000 U/kg feed, more preferably, 250 U/kg to 7500 U/kg, and even more preferably, 500 U/kg to 5000 U/kg. It will be understood that one xylanse U is the amount of enzyme that releases 0.5 μmol of reducing sugar equivalents (as xylose by the DNS [4]) reducing sugar method) from a oat-spelt-xylan substrate per min at pH 5.3 and 50° C. (Bailey, M. J. Biely, P. and Poutanen, K., Journal of Biotechnology, Volume 23, (3), May 1992, 257-270).

It will be understood that the feed supplement may be for any suitable animal. Preferably, the animal is a monogastric animal, for example poultry or swine.

It will be apparent that the feed supplement may contain the at least one phytase and the at least one lipolytic enzyme in any suitable amount. Preferably, the feed supplement comprises at least 0.1% by weight of lipolytic enzyme and phytase either individually or as a combined percentage. More preferably, the feed supplement may comprise at least 0.5%; at least 1%; at least 2%; at least 3%; or at least 4, 5, 6, 7, 8, 9 or 10% by weight of lipolytic enzyme and phytase either individually or as a combined percentage

It will be obvious to the skilled person that the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention may also comprise other components such as stabilising agents and/or bulking agents and/or other enzymes.

Preferably, the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention will be thermally stable to heat treatment up to about 70° C.; up to about 85° C.; or up to about 95° C. The heat treatment may be performed for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term thermally stable means that at least about 75% of the enzyme components that were present/active in the additive before heating to the specified temperature are still present/active after it cools to room temperature. Preferably, at least about 80% of the enzyme components that were present and active in the additive before heating to the specified temperature are still present and active after it cools to room temperature.

The feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention may have a shelf-life of greater than about 30 weeks; greater than about 40 weeks; greater than about 50 weeks; greater than about 1 year; greater than about 1.5 years. The shelf-life means that at least about 80% of the enzyme components that were present and active in the additive when it was prepared are still present and active.

Preferably, the method of preparing a feed supplement according to the present invention comprises a mixing step that comprises admixing the at least one phytase and at least one lipolytic enzyme optionally with at least one physiologically acceptable carrier.

In a particularly preferred embodiment the feed supplement is homogenized to produce a powder.

In an alternative preferred embodiment, the feed supplement is formulated in to granules as described in WO2007/044968 (referred to as TPT granules).

In another preferred embodiments when the feed supplement is formulated into granules the granules comprises a hydrated barrier salt coated over the protein core. The advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other feed additives otherwise having adverse effect on the enzyme.

Preferably, the salt used for the salt coating has a water activity greater than 0.25 or constant humidity greater than 60% at 20° C.

Preferably, the salt coating comprises a Na₂SO₄.

The method of preparing a feed supplement may also comprise the further step of pelleting the powder. The powder may be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strands are cut into suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-100° C., typical temperatures would be 70° C., 85° C., 90° C. or 95° C. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

It will be understood that the feed supplement of the present invention is suitable for addition to any appropriate feed material.

As used herein, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal.

In some embodiments, the feed material will comprise one or more of the following components: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.

It will further be apparent that the feed supplement of the present invention is particularly advantageous when added to a high phytate feed material.

As used herein, the term feedstuff refers to a feed material to which one or more feed supplements have been added.

It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared. It will be further understood that depending on the starting feed material, the feedstuff may be a high fibre feedstuff or a low fibre feedstuff.

Preferably, the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and/or vegetable oils.

Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.

It will be apparent to the skilled person that the feed supplement of the present invention will be particularly beneficial when used in a feedstuff comprising high calcium. More particularly a feedstuff comprising high calcium and high phytate.

It will be apparent to a skilled person that the level of calcium which represents a high calcium diet may vary depending on the type of animal and even the use for which the animal is reared. For example, for laying hens, a diet with >3% would be high Ca diet. For broilers and all turkeys, a diet with >1% Ca would be high Ca diet. For the purposes of the present invention, a high calcium diet is considered to be a diet comprising at least 1-2% calcium.

It will further be apparent to a skilled person that a high phytate diet is one which comprises >0.90% weight of phytic acid.

As defined herein, a low fibre feedstuff is a feedstuff comprising one or more feed materials, which contains a maximum content of water insoluble cell walls of about 25%, and/or a maximum content of soluble non-starch polysaccharides of about 4%. More preferably, a maximum content of water insoluble cell walls of about 22.5%, about 20%, about 17.5%, about 15%, about 12.5%; and/or a maximum content of soluble non-starch polysaccharides of about 3%, about 2.5%, about 2%, about 1.75%, about 1.5%, about 1.25%.

In preferred embodiments, the feed supplement or at least one lipolytic enzyme and at least one phytase according to the present invention is mixed with at least one low fibre feed material, for example, corn, wheat, an animal-by product meal, or soybean and/or any of the by-products to provide a low fibre feedstuff.

Preferably, the feedstuff is a corn soybean meal mix.

Preferably, the feed material is not defatted rice bran.

In preferred embodiments, the feedstuff comprises phytase at a level of about 250 FTU/kg to about 15,000 FTU/kg feedstuff (e.g. 250 to 10,000 FTU/kg, 400-7,500 FTU/kg and also 500-5000 FTU/kg).

In further preferred embodiments, the feedstuff comprises lipolytic enzyme at a level of about 125 LIPU/kg to about 45,000 LIPU/kg feedstuff (e.g. 500 to 30,000 LIPU/kg, 1000-20000 LIPU/kg and also 3000-10000 LIPU/kg).

It will be readily apparent to the skilled person that in order for the feed supplement of the present invention to provide the claimed advantages phytate must be present in the feed material or feedstuff. It will also be readily apparent that this phytate may be present naturally as a constituent of the feed material, or may be added as an additional supplement at a desired level.

In another aspect there is provided a method for producing a feedstuff. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting—in particular by suitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), swine (all age categories), a pet (for example dogs, cats) or fish.

Optionally the feedstuff may comprise further additives. For example, calcium may be added to the feedstuff in any suitable amount to supplement the diet of the animal and/or as a bulking agent. The calcium may be in the form of organic (e.g. leafy green vegetables) or inorganic (e.g. limestone/calcium carbonate) calcium. Preferably, after addition of the calcium supplement, the feedstuff will comprise at least about 0.5% calcium. More preferably, at least about 0.8%, at least about 1.0%, at least about 2.0%, at least about 3% calcium.

The feedstuff may comprise at least 0.0001% by weight of the feed supplement. Suitably, the feedstuff may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500% by weight of the feed supplement.

In a further aspect there is provided the use of at least one phytase and at least one lipolytic enzyme in the manufacture of a feedstuff for increasing the availability of at least one nutrient and/or increasing the available metabolic energy from a feed material.

Preferably, the at least one phytase and at least one lipolytic enzyme are formulated as a feed supplement. More preferably, the feed supplement is the feed supplement according to the present invention.

In preferred embodiments the at least one nutrient is selected from phosphorous; calcium; total fat; and/or amino acids.

As used herein, the term “increasing the availability of at least one nutrient and/or increasing the available metabolic energy” means an increase in the amount of the nutrient or energy available for use by the animal consuming a unit weight the feedstuff compared to the availability of the nutrient or energy available from a unit weight of the feed material to which no phytase and lipolytic enzyme or feed supplement has been added.

In a further preferred embodiment, the feed material is a feed material comprising high levels of phytate and/or calcium.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is further illustrated by reference to the accompanying figures in which:

FIG. 1 shows the amino acid sequence (SEQ ID No. 1) of wild type Buttiauxella phytase.

FIG. 2 shows the amino acid sequence (SEQ ID No. 2) of a variant Buttiauxella phytase designated BP-112.

FIG. 3 shows an amino acid alignment of wild type Buttiauxella phytase and 4 variant Buttiauxella phytases (SEQ ID Nos. 1-5).

FIG. 4 shows the amino acid sequence (SEQ ID No. 6) of a further variant Buttiauxella phytase designated BP-11.

FIG. 5 shows the amino acid sequence (SEQ ID No. 7) of a lipolytic enzyme from Aspergillus tubingensis wherein the endogenous signal peptide is shown in bold.

FIG. 6 shows the amino acid sequence (SEQ ID No. 8) of a lipolytic enzyme from Aspergillus tubingensis which is the same as SEQ ID No. 7 except that the endogenous signal peptide has been removed.

FIG. 7 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No. 7) including the signal sequence—the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ ID No. 9) The signal sequence is shown in bold and the introns are shown in lower case.

FIG. 8 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No. 8) not including the signal sequence—the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ ID No. 10). The introns are shown in lower case.

FIGS. 9A-9B show the resistance of the phytases originating from Buttiauxella, variants BP-17, BP-110, BP-111, and BP-112, and of Phyzyme XP, Natuphos, and Ronozyme P against increasing concentrations of pepsin.

FIG. 10 shows a schematic representation of the pelleting unit at the Technological Institute in Kolding.

FIG. 11 shows the relative residual activity from pelleting trials of three Phytase B variants for use in the present invention.

FIG. 12 shows the relative residual activity when Phyzyme XP, an E. coli phytase is formulated on whole grounded wheat.

FIG. 13 shoes the results of a trial in which the phytase was extracted from the coated Ronozyme product and formulated on whole grounded wheat to study the themostability of the phytase molecule without protection.

FIGS. 14A-14C, 15A-15C, 16A-16C, and 17A-17B show the activity of the phytases for use in the present invention and controls against phytate at various pH's.

FIGS. 18A-18D and 19A-19D are HPLC chromatograms which clearly show that phytase from Buttiauxella variants catalyzed the hydrolysis of phytic acid at temperature greater than 85° C.

FIG. 20 shows the pDONR221::lip 3 containing the Aspergillus tubingensis lipase 3 genomic DNA.

FIG. 21 shows the final lipase expression construct ATlipase3Trex.

FIG. 22 shows the nucleotide sequence (SEQ ID No: 11) encoding wild type Buttiauxella phytase.

FIG. 23A shows Ileal energy digestibility coefficients of cannulated pigs (SEM=0.0009). Bars with different letters differ at a P<0.05 level.

FIG. 23B shows Ileal crude protein digestibility coefficients of cannulated pigs (SEM=0.0009). Bars with different letters differ at a P<0.05 level.

FIG. 24 shows total tract digestible energy coefficients of pigs (SEM=0.009). Bars with different letters differ at a P<0.05 level.

FIG. 25A shows the total tract calcium retention of pigs (SEM=0.027). Bars with different letters differ at a P<0.05 level.

FIG. 25B shows the Total tract phosphorus retention of pigs (SEM=0.030). Bars with different letters differ at a P<0.05 level.

FIG. 26 shows the amino acid sequence of wild type E. coli phytase including the signal sequence (SEQ ID NO: 12).

FIG. 27 shows the amino acid sequence of mature wild type E. coli phytase (SEQ ID NO: 13).

FIG. 28 shows the nucleotide sequence encoding the wild type E. coli phytase (SEQ ID NO:14).

FIG. 29 shows the production of Free Fatty acid from corn-soy feed when incubated with various lipolytic enzyme/phytase combinations.

FIGS. 30A-30B show the production of a phytase resistant complex of phytic acid and calcium and reversal by adding free fatty acids. FIG. 30A shows the effect on phytase activity of increasing CaCl2 concentration. FIG. 30B shows the effect on the phytase activity of adding free fatty acids to the reaction mixture containing 15 mM.

FIGS. 31A-31B show the production of a phytase resistant complex of phytic acid and calcium and reversal by adding Lipase reaction mixture including lipase. FIG. 31A shows the effect on phytase activity of increasing CaCl2 concentration and FIG. 31B shows the effect on the phytase activity of adding Lipase reaction mixture to the phytase reaction mixture containing 30 mM CaCl2.

FIG. 32 shows the pH profile of Lipase 3 and Pancreatic lipase measured in a corn-soy diet.

FIG. 33 shows the ileal digestibility coefficient of phosphorus in broiler chickens. Bars with different letters differ at a P<0.05 level.

FIG. 34 shows the ileal digestibility coefficient of calcium in broiler chickens. Bars with different letters differ at a P<0.05 level.

FIG. 35 shows the total tract retention coefficient of phosphorus in broiler chickens. Bars with different letters differ at a P<0.05 level.

FIG. 36 shows the total tract retention coefficient of calcium in two experiments with broiler chickens. Bars with different letters differ at a P<0.05 level.

FIG. 37 shows the inventors Hypothesis of the mechanism involved in the lipase and phytase interaction in the digestive tract of poultry. The figure shows a schematic representation of the possible mechanism behind the synergistic effect observed between Lipase and phytase. Due to the action of Lipase 3 under acidic conditions, free fatty acids (FFA) are liberated at an earlier stage in digestion compared to the situation without Lipase 3. These FFA may bind with excess calcium (Ca) to form soaps in the gut. There is then less free Ca available to bind to phytic acid (IP6) and form the phytase resistant complex. Eventually, more IP6 is degraded by the phytase as compared to the situation without Lipase 3 added. This leads to increased phosphate (P) liberation and subsequently absorption by the animal. The FFA-Ca complexes may dissolve later in the digestive tract resulting in increased fat and Ca absorption. If no phytase is present to hydrolyse the IP6, this may complex with the FFA-Ca soap to form an insoluble non-degradable IP6-Ca-FFA soap, resulting in decreased fat, Ca, and P absorption.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.

As used herein, the term “homologue” means an entity having a certain homology with the amino acid sequences and the nucleotide sequences. Here, the term “homology” can be equated with “identity”. Suitably, “homologous” in this context refers to the percentage of sequence identity between two enzymes after aligning their sequences using alignment algorithms as described in more detail below.

In the present context, a homologous amino acid sequence is taken to include an amino acid sequence which may be at least 75, 80, 81, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the sequence. Typically, the homologues will comprise the same active sites etc.—e.g. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

By “functional fragment” is meant a fragment of the polypeptide that retains that characteristic properties of that polypeptide. In the context of the present invention, a functional fragment of a phytase enzyme is a fragment that retains the phosphate releasing from phytic acid capability of the whole protein.

For amino acid sequences and nucleotide sequences, homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.

However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.

Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p 387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp P M (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

In a preferable aspect of the present invention the following software and settings for calculating percentage sequence homology/identity are used. For amino acid sequences percentage of identities (homology) or “positives” are calculated by the AlignX Vector NTI (Vector NTI Advance 9.1 from Invitrogen Corporation, Carlsbad, Calif., USA.) for each possible pair of amino acid sequences. Settings are default parameters (Gap opening penalty—10, Gap extension penalty 0.1).

For nucleic acid sequences percentage of identities (homology) or “positives” are calculated by the AlignX Vector NTI programme from Informax Inc. (USA) for each possible pair of nucleic acid sequences. Settings are default settings for DNA are: Gap opening penalty: 15 and Gap extension penalty: 6.66. (same settings for multiple alignments).

Preferably the amino acid identity (homology) is calculated across the full-length amino acid sequence or for nucleic acid to a corresponding polynucleotide which encodes the respective the full-length amino acid sequence.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl Biosci. 9: 745-756) (Taylor W. R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.

Set Sub-set Hydro- F W Y H K M I L V A G C Aromatic F W Y H phobic Aliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E D Positively H K R charged Negatively E D charged Small V C A G S P T N D Tiny A G S

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

The term “protein”, as used herein, includes proteins, polypeptides, and peptides.

The terms “amino acid residue equivalent to”, “amino acid corresponding to” and grammatical equivalents thereof are used herein to refer to an amino acid residue of a protein having the similar position and effect as that indicated in a particular amino acid sequence of a particular protein. The person of skill in the art will recognize the equivalence of specified residues in comparable proteins.

The term “property” or grammatical equivalents thereof in the context of a polypeptide, as used herein, refer to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, temperature and/or pH activity profile, feed processing stability, and ability to be secreted.

As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

The terms “protein” and “polypeptide” are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues are used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

The term “signal sequence” or “signal peptide” refers to any sequence of nucleotides and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein.

By “functional fragment” is meant a fragment of the polypeptide that retains that characteristic properties of that polypeptide. In the context of the present invention, a functional fragment of a phytase or lipolytic enzyme is a fragment that retains the phytase or lipolytic enzyme cleavage capability of the whole protein.

The term “isolated”, “recovered” or “purified” refers to a material that is removed from its original environment. The term “substantially purified” means that the material has been purified to at least a substantial degree.

In one aspect, preferably the nucleotide or amino acid sequence is in an isolated form. The term “isolated” means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene” includes a plurality of such candidate agents and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The enzymes for use in the present invention can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. Culturing is accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of one or more particular microorganism species to be employed.

In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulphur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion. In effect, by using an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced. Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process.

Although the aeration rate can vary over a considerable range, aeration generally is conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, ˜volumes (at the pressure employed and at 25° C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3, volumes (at the pressure employed and at 25° C.) of oxygen per liquid volume in the fermentor per minute.

The pressure employed for the microbial conversion process can range widely. Pressures generally are within the range of about 0 to 50 psig, presently preferably about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved. Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time this is balanced by the fact that high atmospheric pressures do increase equipment and operating costs.

The fermentation temperature can vary somewhat, but for filamentous fungi such as Trichoderma reesei the temperature generally will be within the range of about 20° C. to 40° C., generally preferably in the range of about 25° C. to 34° C., depending on the strain of microorganism chosen.

The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0. With filamentous fungi, the pH normally is within the range of about 2.5 to 8.0; with Trichoderma reesei, the pH normally is within the range of about 3.0 to 7.0. pH range preferences for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art.

While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, generally it will be within the range of about 24 to 500 hours, preferably presently about 24 to 400 hours. Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

Although the fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment. If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermentor. Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermentor, cell density measurable by light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible.

In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121° C. for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though presently preferred is operation under 15 L Biolafitte (Saint-Germain-en-Laye, France).

The collection and purification of the enzymes of the present invention from the fermentation broth can also be done by procedures known per se in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired enzyme product of the present invention, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate. Further purification may optionally be achieved by crystallization or by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.

Phytases

Phytic acid (myo-inositol hexakisphosphate) is an important constituent in cereals, legumes and oilseed crops. The salt form, phytate, is the major storage form of phosphorous in these plants.

As used herein, the term “phytase” or “phytase activity” refers to a protein or polypeptide which is capable of catalyzing the hydrolysis of phytate to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate. For example, enzymes having catalytic activity as defined in Enzyme Commission EC number 3.1.3.8 or EC number 3.1.3.26.

Some phytases in addition to phytate, are capable of hydrolysing at least some of the inositol-phosphates of intermediate degrees of phosphorylation.

The terms “phytase variant” or “variant” or “modified form” refer to a phytase enzyme with an amino acid sequence derived from the amino acid sequence of a parent phytase having one or more amino acid substitutions, insertions, and/or deletions, which together are referred to as “mutations”.

The terms “parent phytase” or “parent enzyme” refer to a phytase enzyme from which a phytase variant is derived. A parent phytase can be a wild type phytase or another phytase variant. In particular, in the present invention, a “parent phytase” may be derived from a Buttiauxella sp. or E. coli as described in WO 99/08539 U.S. Pat. No. 5,876,997, U.S. Pat. No. 6,190,897, and AU 735371 EP 1003379 and JP 2001514869 all of which are incorporated herein by reference. Suitably, the “parent phytase” is derived from Buttiauxella strain P1-29 deposited under accession number NCIMB41248 as described WO2006/043178.

The terms “E. coli phytase” and “Buttiauxella sp. phytase” mean that the enzymes need not have been obtained from a source of E. coli or Buttiauxella sp. Instead, the enzymes preferably have to have the same functional characteristics or sequence as that of the E. coli or Buttiauxella sp. phytase. For example, the Buttiauxella sp. Phytase may be a variant derived from a Buttiauxella sp., but which is not naturally present in Buttiauxella species.

The term “Buttiauxella” refers to a genus of gram negative, facultatively anaerobic bacteria of the family Enterobacteriaceae and Buttiauxella spp include B. agrestis, B. brennerase, B. ferragutiae, B. gaviniae, B. izardii, B. noackiae, and B. warmboldiae. Strains of the Buttiauxella species are available for example from the American Type Culture Collection (ATCC) and DSMZ, the German National Resource Centre for Biological Material.

Buttiauxella phytases are preferably identified from Buttiauxella spp by the methods described in WO 2006/043178.

In an alternative embodiment the parent phytase or phytase may be derived Citrobacter sp. as described in WO2006/038062.

The terms “wild type phytase” or “wild type” as used herein describe a phytase enzyme with an amino acid sequence found in nature. In particular, the wild-type Buttiauxella phytase is that shown as SEQ ID. No. 1. The wild-type E. coli phytase is shown as SEQ ID No. 13 (FIG. 27).

The term “phytase variant” as used herein means a phytase enzyme with an amino acid sequence that differs by at least one amino acid substitution, insertion and/or deletion as compared with the amino acid sequence of the phytase of SEQ. ID NO: 1 or SEQ ID No. 13. The terms “variant” and “variation” as used herein merely means that there is a difference between sequence of the amino acid sequence of the phytase variant and the amino acid sequence of SEQ. ID NO: 1 or 13, and does not mean that an amino acid sequence of SEQ. ID NO: 1 or 13 or any other phytase served as a starting material in any way and/or was physically varied, mutated, modified or otherwise altered to yield the variant. Simply put, the phytase variants for use in the present invention may be prepared by any method, and skilled artisans will be readily familiar with numerous methods, some of which are described herein, for making the phytase variants.

Embodiments of variant phytases (e.g., variant Buttiauxella sp. phytases) for use in the present invention are disclosed in WO 2006/043178, WO 2008/097619 and PCT/US2009/41011 all of which are specifically incorporated herein by reference and which describe phytases obtainable from or derived from a parent Buttiauxella sp. and phytases corresponding to a Buttiauxella sp. phytase enzyme. Specifically, WO 2006/043178 describes the mutagenesis of a wild-type phytase enzyme having the sequence disclosed therein as SEQ ID NO: 3. A number of preferred mutations are taught in WO 2006/043178. PCT/US2009/41011 teaches further preferred mutations. Specific preferred mutations are disclosed herein as SEQ ID NOs: 2-6 these represent:

Phytase BP-11 (sometimes referred to as BP 11)—which is the phytase described in at least PCT application WO 2006/043178 and which may be obtained from Danisco A/S. The amino acid sequence is described herein as SEQ ID NO:6. Phytase BP-17 (sometimes referred to as BP 17)—which is the phytase described in at least PCT application WO 2008/097619 and which may be obtained from Danisco A/S. The amino acid sequence is described herein as SEQ ID NO:3.

BP-110 (sometimes referred to as BP 17 var. 110, or sometimes it is referred to as BP17-110 or BP17 110)—which is the phytase described in at least PCT application PCT/US2009/41011. The amino acid sequence is described herein as SEQ ID NO:4.

BP-111 (sometimes referred to as BP 17 var. 111, or sometimes it is referred to as BP17-111 or BP17 111)—which is the phytase described in at least PCT application PCT/US2009/41011. The amino acid sequence is described herein as SEQ ID NO:5.

BP-112 (sometimes referred to as BP 17 var. 112, or sometimes it is referred to as BP17-112 or BP17 112)—which is the phytase described in at least PCT application PCT/US2009/41011. The amino acid sequence is described herein as SEQ ID NO:2.

The amino acid sequences for BP-11 (SEQ ID NO:6) BP-17 (SEQ ID NO: 3), BP-110 (SEQ ID NO:4), BP-111 (SEQ ID NO:5) and BP-112 (SEQ ID NO:2) are presented in FIG. 3.

When referring to SEQ ID NOs: 1-6 and polypeptides comprising SEQ ID NOs: 1-6, it is envisaged that this also refers to polypeptides that are co- or post-translationally processed during expression, for example by signal peptide cleavage. Post-translational cleavage may also occur at the C-terminal. Therefore in a preferred embodiment the effective fragment thereof (also referred to as functional fragment thereof) is the mature polypeptide produced by the native host or a suitable appropriate expression host.

In another embodiment, the phytase is characterised in that it is derived from Buttiauxella sp. strain P1-29 deposited under accession number NCIMB 41248 as described in WO 2006/043178.

The feed supplements according to the present invention may include phytases having improvements of the characteristics of a parent phytase resulting from modification of one or more amino acid residues of the amino acid sequence of the parent phytase.

Improved phytase enzyme for use in the invention preferably have an identity to SEQ ID NO: 3 or an effective fragment thereof of more than 75%, preferably of more than 80%, more preferably of more than 90%, more preferably of more than 95%, 96%, 97%, 98% preferably of more than 99%. However, it is also envisaged that variants may be heterologous, (i.e. not homologous) to SEQ ID No: 3. For example variants produced by recombination techniques such as exo-mediated recombination, or family shuffling, may result in variants, although prepared using the parent phytase, may have less than 75% homology.

Suitably the variants show enhanced stability characteristics with respect to any one of the following: thermal stability, thermal activity, pH range, pepsin stability, specific activity, substrate specificity, and broader substrate specificity. Suitable methods for determining these characteristics are disclosed herein.

The term “enhanced stability” in the context of a property such as stability at higher temperatures, lower pH, etc. refers to a higher retained enzyme activity over time as compared to another identified phytase such as that of, in the case of Buttiauxella, SEQ ID NO: 1. Unless another phytase is specifically identified, the term “enhanced stability” when used herein, will refer to a higher retained enzyme activity over time as compared to the phytase of SEQ ID NO: 1.

The terms “thermally stable” and “thermostable” refer to phytases of the present disclosure that retain a specified amount of enzymatic activity after exposure to an elevated temperature. Phytase variants according to this disclosure are considered thermostable at a specified temperature if the enzyme retains greater than 50% of its activity after exposure to the specified temperature for 10 minutes at pH 5.5 in buffer.

The term “enhanced thermal activity” as it pertains to phytase variants of the present disclosure means that the phytase variant exhibits the same or an increased amount of phytase enzyme activity at elevated temperature as compared to the phytase enzyme activity of another identified phytase such as that of SEQ ID NO: 1. Unless another phytase is specifically identified, the term “improved thermal activity” when used herein will refer to the thermal activity of a phytase variant of this disclosure as compared to the thermal activity of the phytase of SEQ ID NO: 1.

Variants of this disclosure as described in WO 2006/043178, WO 2008/097619 and PCT/US2009/41011, the disclosure of which is incorporated herein by reference, are described by the following nomenclature: [original amino acid residue of SEQ. ID NO: 1/position of original amino acid residue in SEQ. ID NO: 1/substituted amino acid residue]. For example, in SEQ ID NO: 2, the substitution of threonine (T) for the original alanine (A) in position 89 of SEQ ID NO: 1 is represented as A89T. When a position suitable for substitution is identified herein without a specific amino acid suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Where a variant phytase contains a deletion in comparison with other phytases the deletion is indicated with “*”. For example, a deletion at position A89 of SEQ. ID NO: 1 is represented as A89*. A deletion of two or more consecutive amino acids is indicated, for example, as (89-91)*.

The terms “derived from” and “obtained from” refer to not only a phytase produced or producible by a strain of the organism in question, but also a phytase encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the terms refers to a phytase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the phytase in question. Hence, a phytase that is “derived from” and “obtained from” another phytase does not necessarily mean that the phytase has been physically derived or physically obtained from the second phytase, but rather can also mean that the phytase in question has been prepared using knowledge or ideas derived from knowledge of the second phytase.

In particular, the improvements in phytase characteristics are directed to the enzyme stability under food and feed processing conditions, to the enzyme stability during stomach transit, and to the enzyme activity and stability in human or animal stomach and/or intestinal tract making the improved variants particularly suitable for use as feed supplements. Thus, such improvements comprise among other parameters the increase in stability at elevated temperatures, preferably at temperatures above 65° C., the increase in stability against proteolytic digestion, preferably protease of the digestive tract such as pepsin, the increase in catalytic activity at low pH, preferably catalytic activity below pH 5.5, and the general efficiency of releasing phosphate groups from phytate, and preferably in addition inositol phosphates.

Therefore, in some embodiments the invention relates to feed supplements comprising phytase variants with improved characteristics that, when compared to the parent phytase, comprise mutations at one or more of the positions disclosed in WO 2006/043178, WO 2008/097619 and PCT/US2009/41011 and/or at corresponding positions in a phytase homologous to the phytase as shown in the amino acid sequence of Seq ID No 1. These positions are characterized in that mutagenesis of these positions lead to an improvement in the enzymes characteristics.

Variants with higher thermal stability (thermal stability difference) are preferably determined using the methods disclosed in WO 2006/043178.

The variant phytase enzyme for use in the present invention preferably has a thermal stability difference (T.D) of at least 1.5, more preferably 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 most preferably at least 20.

Variants with higher proteolytic stability are preferably determined by the methods disclosed in WO 2006/043178.

Preferably the phytase enzyme variant for use in the present invention has a proteolytic stability (residual activity) of at least 45%, preferably 50%, 55%, more preferably at least 60% or 65%, most preferably at least 70%.

Preferably the phytase variant for use in the present invention has a specific activity of greater than 100% of wt activity at pH 4.0, preferably greater than 105%, 110%, more preferably greater than 114%.

In one aspect, preferably the amino acid sequence is in a purified form. The term “purified” means that the sequence is in a relatively pure state at least 1%, 5% pure or 10% pure, more preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% pure. In a preferred embodiment, when referring to a polypeptide, the purity as defined above is determined in terms of being purified from other polypeptides by SDS-PAGE electrophoresis.

Variants/Derivatives

The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme or of any nucleotide sequence encoding such an enzyme.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

Other Components

The feed supplement of the present invention may be used in combination with other components or carriers.

Suitable carriers for feed enzymes include maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, anti-foam, Na2SO4, Talc, PVA and mixtures thereof. In addition there are a number of encapsulation techniques including those based on fat/wax coverage, adding plant gums etc.

Examples of other components include one or more of: thickeners, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles, excipients, diluents, lubricating agents, flavouring agents, colouring matter, suspending agents, disintegrants, granulation binders etc. These other components may be natural. These other components may be prepared by use of chemical and/or enzymatic techniques.

As used herein the term “thickener or gelling agent” as used herein refers to a product that prevents separation by slowing or preventing the movement of particles, either droplets of immiscible liquids, air or insoluble solids.

The term “stabiliser” as used here is defined as an ingredient or combination of ingredients that keeps a product (e.g. a food product) from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. a food product ingredient) that prevents the separation of emulsions.

As used herein the term “binder” refers to an ingredient (e.g. a food ingredient) that binds the product together through a physical or chemical reaction.

The term “crystal modifier” as used herein refers to an ingredient (e.g. a food ingredient) that affects the crystallisation of either fat or water.

“Carriers” or “vehicles” mean materials suitable for compound administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubiliser, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

Examples of nutritionally acceptable carriers include, for example, grain, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, and the like.

Examples of excipients include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesium stearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are in admixture together or even when they are delivered by different routes) or sequentially (e.g. they may be delivered by different routes).

As used herein the term “component suitable for animal or human consumption” means a compound which is or can be added to the composition of the present invention as a supplement which may be of nutritional benefit, a fibre substitute or have a generally beneficial effect to the consumer.

By way of example, the components may be prebiotics such as alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), lactosucrose, soybean oligosaccharides, palatinose, isomalto-oligosaccharides, gluco-oligosaccharides and xylo-oligosaccharides.

Lipolytic Enzymes

Lipolytic enzymes (EC 3.1.1.3), which can be defined as carboxylesterases which catalyze the hydrolysis of acylglycerols, are physiologically very important enzymes as one of the three major digestive enzymes together with amylases and proteases. They hydrolyse lipids to glycerol and fatty acids, but can also function in esterification or transesterification reactions.

In preferred embodiments, the lipolytic enzyme for use in the present invention is a lipolytic enzyme derived from the filamentous fungus Aspergillus tubingensis as disclosed in WO 98/45453 and deposited under the Budapest Treaty with the NCIMB on 24 Feb. 1997 under the accession number 40863. It will be understood that the lipolytic enzyme may be derived from Aspergillus, or alternatively may be recombinantly produced in another organism such as E. Coli.

Preferably, the lipolytic enzyme comprises a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 7 or 8 or a polypeptide having at least 70%, 80%, 90%, 95%, 98% or 99% identity thereto; or a polypeptide which is produced by expression of a nucleotide sequence comprising the sequence of SEQ ID NO: 9 or 10; or a sequence which differs from SEQ ID NO:9 or 10 due to the degeneracy of the genetic code; or a sequence which has at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 9 or 10.

It will be understood that any general definitions of features disclosed in relation to phytases can be equally applied to lipolytic enzymes and, for the sake of conciseness will not be repeated here.

In a further preferred embodiment, the lipolytic enzyme for use in the present invention is produced in a Trichoderma reesei cell as disclosed herein.

The method of producing the lipolytic enzyme comprising the steps of:

-   -   (i) providing a Trichoderma reesei cell comprising         -   a) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme comprising an amino acid sequence shown as             SEQ ID NO: 7 or SEQ ID NO: 8 or an amino acid sequence which             has at least 70% sequence identity to SEQ ID NO: 7 or 8;             and/or         -   b) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises             the nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO:             10 or a nucleotide sequence which has at least 70% sequence             identity to SEQ ID NO: 9 or SEQ ID NO: 10; and/or         -   c) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises a             nucleotide sequence which hybridizes to SEQ ID NO: 9 or SEQ             ID NO: 10 or a nucleotide sequence which has at least 70%             sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10 or the             complement of any thereof under stringent conditions; and/or         -   d) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence is             identical to the nucleotide sequence shown as SEQ ID NO: 9             or SEQ ID NO: 10 except for the degeneracy of the genetic             code; and     -   (ii) culturing the cell under conditions to allow for expression         of said heterologous nucleotide sequence(s) encoding said         lipolytic enzyme.

It will be understood that as defined herein, the term stringent conditions refers to washing at. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015 M Na3citrate pH 7.0}.

It will be understood that these conditions may also be high stringent conditions which are defined herein as washing at 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na3citrate pH 7.0}.

Alternatively there is provided a method of producing a lipolytic enzyme comprising the steps of:

-   -   (i) transfecting or transforming a Trichoderma reesei cell with         -   a) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme comprising an amino acid sequence shown as             SEQ ID NO: 7 or SEQ ID NO: 8 or an amino acid sequence which             has at least 70% sequence identity to SEQ ID NO: 7 or 8;             and/or         -   b) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises             the nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO:             10 or a nucleotide sequence which has at least 70% sequence             identity to SEQ ID NO: 9 or SEQ ID NO: 10; and/or         -   c) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises a             nucleotide sequence which hybridizes to SEQ ID NO: 9 or SEQ             ID NO: 10 or a nucleotide sequence which has at least 70%             sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10 or the             complement of any thereof under stringent conditions; and/or         -   d) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence is             identical to the nucleotide sequence shown as SEQ ID NO: 9             or SEQ ID NO: 10 except for the degeneracy of the genetic             code; and     -   (ii) culturing the cell under conditions to allow for expression         of said heterologous nucleotide sequence(s) encoding said         lipolytic enzyme.

Further there is provided a method of producing a lipolytic enzyme comprising the steps of:

-   -   (i) transfecting or transforming a Trichoderma reesei cell with         -   a) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme comprising an amino acid sequence shown as             SEQ ID NO: 7 or SEQ ID NO: 8 or an amino acid sequence which             has at least 70% sequence identity to SEQ ID NO: 7 or 8;             and/or         -   b) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises             the nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO:             10 or a nucleotide sequence which has at least 70% sequence             identity to SEQ ID NO: 9 or SEQ ID NO: 10; and/or         -   c) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence comprises a             nucleotide sequence which hybridizes to SEQ ID NO: 9 or SEQ             ID NO: 10 or a nucleotide sequence which has at least 70%             sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10 or the             complement of any thereof under stringent conditions; and/or         -   d) at least one heterologous nucleotide sequence encoding a             lipolytic enzyme wherein the nucleotide sequence is             identical to the nucleotide sequence shown as SEQ ID NO: 9             or SEQ ID NO: 10 except for the degeneracy of the genetic             code; and     -   (ii) repeating step (i) on the cell to sequentially transfect or         transform the cell with at least one additional heterologous         nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c) (e.g.         such as a heterologous nucleotide sequence encoding a lipolytic         enzyme comprising an amino acid sequence shown as SEQ ID NO: 7         or SEQ ID NO: 8 or an amino acid sequence which has at least 40%         sequence identity to SEQ ID NO: 7 or 8); and     -   (iii) culturing the cell under conditions to allow for         expression of said heterologous nucleotide sequence(s) encoding         said lipolytic enzyme.

Trichoderma reesei is capable of producing the lipolytic enzymes in significantly high yields.

Furthermore, the lipolytic enzymes produced by the methodology disclosed herein are not over glycosylated and therefore have good enzyme activity.

Bradner et al. (in Current Genetics, 44: 224-230, (2003)) used Trichoderma reesei as an expression host organism in their search for previously unknown lipolytic enzymes. A lipolytic enzyme from an Antarctic isolate of Penicillium allii was cloned and expressed in Trichoderma reesei. Bradner et al. concluded that the methods described would be useful for prospecting for potentially novel lipolytic enzyme genes but no suggestion was made that T. reesei could be used for over expression of proteins.

Also disclosed in another aspect is a method of producing a lipolytic enzyme comprising the steps of:

(i) providing a transformed or transfected Trichoderma reesei cell comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme;

(ii) culturing the cell at a pH 4 to pH 5.5 under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme;

(iii) isolating, purifying or concentrating the enzyme in a medium at pH 5.5 to pH 6.5.

In this aspect, preferably the lipolytic enzyme:

a) comprises an amino acid sequence shown as SEQ ID NO: 7 or SEQ ID NO: 8 or comprises an amino acid sequence which has at least 70% sequence identity to SEQ ID NO: 7 or 8; and/or

b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO: 9 or SEQ ID NO: 10 or comprising a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10; and/or

c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 9 or SEQ ID NO: 10 or comprises a nucleotide sequence which is at least 70% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10 or the complement of any thereof under stringent conditions; and/or

d) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence is identical to the nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO: 10 except for the degeneracy of the genetic code.

Suitably there may be at least one heterologous nucleotide sequence encoding the lipolytic enzyme in the Trichoderma reesei cell. There may be two or more copies (i.e. multiple copies) of the or each heterologous nucleotide sequence encoding the lipolytic enzyme according to the present invention in the Trichoderma reesei cell. Each heterologous nucleotide sequence is associated with and is under the control of a promoter. Suitably each heterologous nucleotide sequence may have a separate promoter associated with it and be under the control of that promoter. The promoters may be the same or different.

Thus, the Trichoderma reesei cell may comprise or be transfected or transformed with at least 2 heterologous nucleotide sequences encoding the lipolytic enzyme.

Suitably, the (or each) heterologous nucleotide sequence may comprise a nucleotide sequence which encodes a signal peptide, which nucleotide sequence encoding said signal peptide is operably linked to said nucleotide sequence encoding said lipolytic enzyme. If there are multiple heterologous nucleotide sequences and wherein more than one has a signal sequence associated therewith, then the signal sequences may be the same or different.

Suitably the lipolytic enzyme may comprise an endogenous or exogenous signal peptide. When the signal peptide is endogenous—it means that the signal peptide is that which is naturally linked with the lipolytic enzyme when produced naturally. For example, the signal peptide may be the signal peptide in Aspergillus tubingensis which is naturally linked with the lipolytic enzyme when found in Aspergillus tubingensis.

The term “heterologous” as used herein means that it is does not occur naturally in the Trichoderma reesei cell. In other words, it is exogenous to the Trichoderma reesei cell. For example the term “heterologous nucleotide sequence” as used herein means that the nucleotide sequence does not occur naturally in the Trichoderma reesei cell. In other words, the nucleotide sequence is exogenous to the Trichoderma reesei cell. The term also includes multiple copies of the naturally occurring sequence as such additional multiple copies would be heterologous.

The heterologous nucleotide sequence may be obtained or obtainable from a microorganism, particularly a fungi.

The heterologous nucleotide sequence may be obtained or obtainable from Aspergillus, particularly Aspergillus tubingensis.

In preferred embodiments of the above aspects, said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.

Yet further provided is a lipolytic enzyme obtainable by the methods of the present invention.

Also provided is a transformed or transfected Trichoderma reesei cell comprising:

-   -   a) at least one heterologous nucleotide sequence encoding a         lipolytic enzyme protein having at least 70% sequence identity         to SEQ ID NO: 7 or 8; and/or     -   b) at least one heterologous nucleotide sequence encoding a         lipolytic enzyme wherein the nucleotide sequence comprises the         nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO: 10 or a         nucleotide sequence which has at least 70% sequence identity to         SEQ ID NO: 9 or SEQ ID NO: 10; and/or     -   c) at least one heterologous nucleotide sequence encoding a         lipolytic enzyme wherein the nucleotide sequence comprises a         nucleotide sequence which hybridizes to SEQ ID NO: 9 or SEQ ID         NO: 10 or a nucleotide sequence which is at least 70% sequence         identity to SEQ ID NO: 9 or SEQ ID NO: 10 or the complement of         any thereof under stringent conditions; and/or     -   d) at least one heterologous nucleotide sequence encoding a         lipolytic enzyme wherein the nucleotide sequence is identical to         the nucleotide sequence shown as SEQ ID NO: 9 or SEQ ID NO: 10         except for the degeneracy of the genetic code; and     -   wherein said Trichoderma reesei cell has at least two genes         encoding non-lipolytic enzymes suppressed.

Preferably, said Trichoderma reesei cell according to any of the preceding aspects has at least three genes encoding non-lipolytic enzymes suppressed.

Preferably, said Trichoderma reesei cell according to any of the preceding aspects has at least four genes encoding non-lipolytic enzymes suppressed.

“Suppressed” means that the cell does not express the relevant non-lipolytic enzyme at the same level as the non-transformed/transfected cell. In some embodiments, “suppressed” means that the cell does not express the relevant non-lipolytic enzyme. The suppression may be brought about by techniques known in the art, such as by deletions.

Preferably, at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a cellulase gene.

Preferably, at least two of the genes encoding a non-lipolytic enzyme that is suppressed are cellulase genes.

An example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding a cellobiohydrolases (e.g. CBHI or CBHII).

Another example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding an endoglucanases (e.g. EGI and EGII).

In some embodiments the Trichoderma reesei cell is a cell in which the endogenous genes encoding one or both cellobiohydrolases (CBHI and CBHII) and/or one or both of the endoglucanases (EGI and EGII) are deleted or disrupted. Suitably the Trichoderma reesei cell may be a non-GMM cell or derivative thereof, for example a derivative of the strain RL-P37. Suitably the Trichoderma reesei cell may be a derivative of the strain RL-P37 that is produced using the method set out in Example 7.

In some embodiments, the heterologous nucleotide sequence may encode a lipolytic enzyme comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NO: 7 or SEQ ID No. 8.

In some embodiments the heterologous nucleotide sequence may encode a lipolytic enzyme and comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NO: 9 or SEQ ID No. 10.

Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NO: 7 or SEQ ID No. 8.

Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme and comprises a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NO: 9 or SEQ ID No. 10.

The amino acid sequence identity of a number of lipolytic enzymes to the lipolytic enzyme from Aspergillus tubingensis having the sequence shown in SEQ ID NO: 9 (also called herein lipase 3) are shown in Table 1 below.

TABLE 1 Lipolytic enzymes with sequence identity to the lipolytic enzyme from Aspergillus tubingensis (lipase 3). Amino acid Lipolytic enzymes from sequence different fungi Accession No. identity % A. niger CBS 513.88 XP_001397501.1 93 Aspergillus niger ABG73613.1 93 Aspergillus niger ABG37906.1| 93 Aspergillus nidulans FGSCA4 XP_681315.1 61 Aspergillus clavatus NRRL 1 XP_001276337.1 57 Neosartorya fischeri NRRL 181 XP_001266329.1 60 Aspergillus fumigatus Af293 XP_748138.1 59 Aspergillus oryzae RIB40 XP_001818694.1 56 Aspergillus terreus NIH2624 XP_001218444.1 54 Penicillium chrysogenum CAP96359.1 55 Wisconsin 54-1255 Aspergillus niger ABG73614.1 54 Aspergillus niger XP_001393532.1 53 Thermomyces O59952.1 50 lanuginosus Penicillium marneffei ATCC XP_002147144.1 49 18224 Aspergillus oryzae R XP_001824529.1 44 Phaeosphaeria nodorum SN15 XP_001796872.1 45 Penicillium cyclopium P61869.1 42 Penicillium camemberti. 1TIA_A. 42

The Trichoderma reesei host cell may be any Trichoderma reesei cell. The cell may be considered to be a wild type Trichoderma reesei cell. The Trichoderma reesei cell may be one from which genes encoding one or more secreted cellobiohydrolases (CBHI or CBHII) has/have been deleted or disrupted so that they are not expressed. Suitably the Trichoderma reesei cell may be a non-genetically modified cell or derivative thereof, for example a derivative of the strain RL-P37. Suitably the Trichoderma reesei cell may be a derivative of the strain RL-P37 that is produced using the method set out in Example 10.

The enzyme may be produced in a fermentor which may comprise about 3 liters to about 20 liters culture media. In another embodiment the present invention is carried out as a 10-16 liter, preferably a 14 liter scale fermentation. In one embodiment, preferably the fermentation is carried out with more than about 12 liters, preferably more than about 14 liters.

The Trichoderma reesei host cell is preferably suitable for use in large scale fermentation.

In preferred embodiments, lipolytic enzyme production is carried out on a commercial scale. In this respect, the fermentation is carried out at more than about 50,000 liters scale, preferably more than about 80,000 liters scale, preferably at more than about 200,000 liter scale fermentation.

In one embodiment the total protein produced by the method is well in excess of about 20 g/liter.

Of the total protein produced the majority is the desired lipolytic enzyme. In one embodiment the total secreted protein produced comprises at least 50% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced comprises at least 60% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced comprises at least 70% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced comprises at least 80% of the desired lipolytic enzyme.

Suitably the method may comprise selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected).

In one embodiment suitably the method may comprise a first transformation step wherein a Trichoderma reesei cell is transformed with at least one heterologous nucleotide sequence encoding the lipolytic enzyme defined herein, selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected), and a second transformation step (i.e. retransformation step) of a selected transformant with at least one heterologous nucleotide sequence encoding the lipolytic

enzyme defined herein, followed by further selection of new transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected).

In some embodiments the lipolytic enzyme for use in the present invention maybe glycosylated. In some embodiments, the lipolytic enzyme may be N-glycosylated at N32 (when numbered using SEQ ID NO: 8) or at an equivalent position for other lipolytic enzymes according to the invention. This aspect may impart significant advantages in that the activity of the enzyme is not disrupted or reduced by glycosylation of the enzyme. Without wishing to be bound by theory, reduction in activity of the lipolytic enzyme can be seen when it is produced in other hosts such as A. tubingensis and is thought to be due to over-glycosylation of the enzyme at at least the N242 site.

The lipolytic enzyme produced by the method disclosed herein is therefore distinguishable from the lipolytic enzyme produced in other hosts, e.g. A. tubingensis, because of the degree of glycosylation of the enzyme, particularly at the N32 site. In some embodiments of the present invention the enzyme has glycosylation at, at least, the N32 site.

Suitably the lipolytic enzyme may be produced with a signal peptide. In other words the heterologous nucleotide sequence used in the present invention comprises a portion thereof which encodes a signal peptide.

The signal peptide may be used to direct secretion of the lipolytic enzyme through a particular cell membrane. The signal peptide sequences may be endogenous or exogenous to the lipolytic enzyme coding sequence. For instance, the signal peptide may be the signal peptide which is endogenous to the lipolytic enzyme is Aspergillus tubingensis. Alternatively, the coding sequence for the signal peptide may be obtained (or obtainable) from a cellobiohydrolase gene of Trichoderma reesei.

However, any signal peptide coding sequence capable of directing the expressed lipolytic enzyme into the secretory pathway of a Trichoderma reesei cell of choice may be used.

When we refer to improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield this is compared with conventional methods of expressing this lipolytic enzyme. For example, there is provided an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield compared with production of the lipolytic enzyme in another host organism (i.e. a host organism other than T. reesei). In particular, there is an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield of the lipolytic enzyme by the present invention (i.e. produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in WO98/45453, as incorporated herein by reference).

The term “improved glycosylation” as used herein means that, preferably, glycosylation occurs at N32 (when numbered using SEQ ID NO: 8). Without wishing to be bound by theory, in some situations, the lipolytic enzyme produced in host cells other than T. reesei (and particularly in Aspergillus tubingensis (e.g. as taught in WO98/45453)) may be glycosylated (or overglycosylated), particularly at N242. Therefore, the lipolytic enzyme produced in host cells other than T. reesei and particularly in Aspergillus tubingensis (e.g. as taught in WO98/45453) may be glycosylated at both N32 and N242 sites. However, and without wishing to be bound by theory, the N242 site is in the vicinity of one of the active site residues, namely His258 of SEQ ID No. 8. Thus, it is believed that glycosylation (or overglycosylation) at the N242 site can lead to a reduced activity (i.e. lipase activity) of the enzyme. The lipolytic enzyme of the present invention does not have reduced activity. Glycosylation of the lipolytic enzyme of the present invention may occur at the N32 site which is away from the active site residues, such as His258.

The term “improved enzyme activity” as used herein means that the activity is the same as or greater than the lipase activity of the lipolytic enzyme produced naturally by Aspergillus tubingensis.

By enzyme activity we mean at least lipase activity. Enzyme activity (e.g. lipase activity) can be measured using the relevant protocols set out below in the Examples section.

It has been surprisingly found that the lipolytic enzyme produced in accordance with the method disclosed herein is easy to isolate from the medium into which it has been excreted—i.e. the culture (fermentation) broth—as high expression levels are obtained.

Thus, the method may involve one or more of the following steps to the medium into which the lipolytic enzyme has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free); granulating said medium (preferably wherein said medium is cell-free).

The method may also involve the following steps to the medium into which the enzyme has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free); and optionally granulating said medium (preferably wherein said medium is cell-free).

The method also optionally involves undertaking the following steps to the medium into which the enzyme has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free); and granulating said medium (preferably wherein said medium is cell-free).

Preferably, the method involves the following steps to the medium into which the enzyme has been secreted following culturing of the cell: diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and optionally granulating said medium wherein said medium is cell-free.

In a preferred aspect, the method involves the following steps to the medium into which the enzyme has been secreted following culturing of the cell: diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and granulating said medium wherein said medium is cell-free.

Preferably the lipolytic enzyme precipitates out of solution in the fermentation broth. Preferably, the lipolytic enzyme precipitate is re-solubilised by pH adjustment. Preferably the pH is adjusted to a pH above the pH of the fermentation broth.

In addition to the advantages mentioned above, another advantage of the enzyme produced by the method is that it enables commercial scale production of the lipolytic enzyme. The method allows for the lipolytic enzyme to be produced in a high yield.

One advantage of the lipolytic enzyme disclosed herein is that it has surprisingly been found that it is possible to go directly from the transformation and screening step (i.e. say from the microtitre plate) directly to large scale fermentation (e.g. at least 14 liter fermentation). This is surprisingly possible because the screening step (particularly the microtitre plate results) are highly predictive of good performance in the large scale fermentation. This contrasts with conventional methods where it is often necessary to cultivate the strain in flasks before moving to larger scale fermentation) This has significant advantages in shortening the production time and/or simplifying the overall procedure and/or reducing costs.

A further advantage is that the methods described herein provide an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with conventional methods of expressing this lipolytic enzyme. For example, there is provided an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with production of the lipolytic enzyme in another host organism (i.e. a host organism other than T. reesei). In particular, there is an increased expression and/or an improved yield of the lipolytic enzyme (i.e. produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in WO98/45453, as incorporated herein by reference).

A further advantage is that the lipolytic enzyme produced in accordance with the methods disclosed herein is easy to produce and isolate and/or purify and/or concentrate.

A further advantage is that the lipolytic enzyme produced in accordance with the disclosed method is easy to re-solubilise.

A further advantage is that the lipolytic enzyme produced in accordance with the disclosed method may be used as a granulate or as a solution.

Lipolytic Enzyme

The term “lipolytic enzyme” as used herein means an enzyme with triacylglycerol hydrolysing activity (classified as E.C. 3.1.1.3).

Suitably, the lipolytic enzyme for use in the present invention may exhibit one or more of the following additional activities: glycolipase activity (E.C. 3.1.1.26), phospholipase A2 activity (E.C. 3.1.1.4), phospholipase A1 activity (E.C. 3.1.1.32) or phospholipase B activity (E.C. 3.1.1.5). The term “glycolipase activity” as used herein encompasses “galactolipase activity”.

Isolated

In one aspect, preferably the lipolytic enzyme for use in the present invention is in an isolated form. The term “isolated” means that the lipolytic enzyme is at least substantially free from at least one other component with which the lipolytic enzyme is naturally associated in nature and as found in nature. The term “isolated” may mean that the lipolytic enzyme is at least substantially free from at least one other component in the culture media in which it is produced. The lipolytic enzyme of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated or with which the enzyme may be produced.

Thus, for example it may be substantially free of the cell(s) or one or more potentially contaminating polypeptides and/or nucleic acid molecules. The lipolytic enzyme may be isolated by separating the cell(s) from the broth during or after fermentation so that the lipolytic enzyme remains in the broth. The lipolytic enzyme may be isolated by subjecting the fermentation broth to cell separation by vacuum filtration.

Purified

In one aspect, preferably the lipolytic enzyme for use in the present invention is in a purified form. The term “purified” means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.

Concentrate

In one aspect, preferably the lipolytic enzyme for use in the present invention is used as a concentrate. The concentrate may be a concentrated form of the medium into which the enzyme has been excreted. Preferably, the concentrate may be a concentrated form of the medium into which the enzyme has been secreted and wherein the cell(s) have been removed.

Materials and Methods

1. Lipase Assay at pH 5.5

As used herein, 1 LIPU (lipase unit) is defined as the amount of enzyme which releases 1 μmol of H⁺ per minute under the conditions described herein below.

5% (v/v) tributyrin substrate is prepared by Mixing 15.00 ml tributyrin, 50.00 ml emulsifying agent and 235 ml dist. water for 20 sec on a homogenizer. The pH of the substrate is adjusted to approx. 5.4 with 0.5 M NaOH.

Emulsifying agent is prepared by mixing 17.9 g NaCl, 0.41 g KH₂PO₄, 400 ml dist. water, and 450 ml glycerol in a 2000 ml beaker. Under vigorous stiffing add 6.0 g Gum Arabic and continue stirring until gum Arabic is completely dissolved. Transfer the solution to a 1000 ml volumetric flask and fill to the mark with dist water.

For dry samples: in a volumetric flask dissolve an amount of enzyme calculated to give a final solution of approximately 3.5 LIPU/ml in half of the final dilution and subject to magnetic stiffing for 20 minutes

After stiffing, adjust to final dilution with distilled water. Any further dilution should be made with distilled water. Samples in solution are diluted directly in distilled water

-   -   25.00 mL of substrate is adjusted to 30.0° C.     -   Adjust substrate pH to 5.50 with NaOH/HCl     -   While stirring, add 2.00 mL sample, and initiate immediately         pH-stat titrator.     -   Stop titration after 6 minutes.     -   Calculate slope of the titration curve. The slope of the         titration curve is calculated from data between 3 and 6 min. The         slope must be in the interval 0.1-0.2 mL/min.     -   The activity (LIPU/g) of the enzyme is calculated using the         following:

${{LIPU}\text{/}g} = \frac{m\;{1/{\min.}} \times N \times 1000 \times F \times {factor}\mspace{14mu}{for}\mspace{14mu}{tributyrin}}{A \times 2}$

-   -   ml/min.: Slope of titration curve     -   N: Normality of NaOH     -   F: Dilution of sample     -   A: Gram sample weighed     -   2: ml sample         2. Lipase Assay at pH 3.5

1 LPLU (low pH lipase unit) is defined as the quantity of enzyme that produces 1 microequivalent of free fatty acid per min under the conditions described herein below.

Trioctanoate substrate: 0.15% Trioctanoate glycerol, 13% Triton X-100, 0.3% NaCl, and 120 mM Glycine-HCl, pH 3.5. The substrates were prepared by mixing all components followed by stiffing for one hour.

The enzyme sample solution is diluted in 50 mM Glycine-HCl, pH 3.5

The assay is carried out using a Konelab analysis robot (Thermo, Vantaa, Finland). 50 trioctanoate substrate is equilibrated at 30° C. for 3 min before mixed with 10 μl enzyme sample solution, and incubation for 6 min at 30° C. Free fatty acid in the reaction mixture was measured using the NEFA-HR(2) kit (WAKO Chemicals GmbH). 110 μl NEFA reagent R1 is added to the reaction mixture and incubated for 3 min at 30° C. before 55 μl NEFA reagent R2 is added. Incubation is continued for another 4.5 min at 30° C. before OD_(520 nm) is measured. The content of free fatty acid is calculated from a standard curve prepared from NEFA Standard (WAKO Chemicals GmbH).

1 LPLU corresponds to 0.24 LIPU for Aspergillus tubingensis lipase (FIG. 5, SEQ ID No. 7).

2. Phytase Assay.

As used herein 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined herein

Principle:

The Phytase is incubated with Sodium phytate, which results in the release of in-organic Phosphate. The in-organic Phosphate creates coloured complexes with the Molybdate-Vanadate reagent, which hereby turns yellow. The yellow colour of the complex is measured on the spectrophotometer at 415 nm.

Quantification of activity is made by an absolute method using a phosphate standard curve.

Reagents:

1.1 Acetate buffer (pH 5.5) 0.25 M 1000 mL

Dissolve:

-   -   30.02 g Sodiumacetate—Trihydrate (18.10 g         sodiumacetate—Anhydrate),     -   0.147 g Calcium chloride—Dihydrate,     -   1.76 g 100% Acetic acid (=1.677 mL, density=1.0498 g/mL) in app.         900 mL water,     -   Adjust pH to 5.5 with 4 M Acetic acid (22.9 mL concentrated         Acetic acid in 100.0 mL deionised water) and transfer the         solution to a 1000 mL volumetric flask.     -   Add 1 mL 10% Tween 20 and adjust the solution to 1000 mL by         addition of deionised water.         1.2 Potassium dihydrogen phosphate 18 mM 1000 mL

Stock solution used for standard curve

-   -   Dry KH₂PO₄ at 60° C. overnight in a heating cupboard.     -   Weigh 2.45 g dry KH₂PO₄ and dilute in Acetate buffer (1.1).         Adjust to 1000.0 mL.         1.3 Phytate solution (substrate) 7.5 mM 250 mL

Dissolve:

-   -   2.10 g (±0.05 g) Sodium phytate, deca hydrate (Phytic acid) in         200 mL Acetate buffer.     -   Thermostat the solution to 37° C. in a water bath.     -   At 37° C. pH is adjusted to pH 5.5 by addition of 4 M Acetic         acid (24 g=22.9 mL concentrated Acetic acid in 100.0 mL         deionised water).     -   Adjust to 250.0 mL by addition of Acetate buffer.     -   A substrate correction factor may be needed when changing to         another lot of phytic acid.     -   Correction factor for phytic acid Sigma P0109, batch 057K0049 is         defined to 1,00         1.4 Nitric acid solution 24.5% 200 mL

(For Ammonium Vanadate solution)

-   -   75 mL Nitric acid (65%) is added to 100 mL water under         continuous stirring.     -   The solution is transferred to a 200 mL volumetric flask and         adjusted with deionised water.         1.5 Ammonium solution 25% 50 mL

(For Ammonium Heptamolybdate solution)

-   -   40 mL of a 32% Ammonium solution is adjusted to 50 mL with         water.         1.6 Ammonium Heptamolybdate solution 10% 250 mL

(For Colour/Stop reagent)

Dissolve:

-   -   25 g Ammoniumheptamolybdate—tetrahydrate in 225 mL water. The         solution needs to be heated to dissolve the Ammonium         Heptamolybdate before adding the Ammonium solution.     -   Add 2.5 mL Ammonium solution (25%) (see 1.5), turn off the heat         and leave on stirrer until totally dissolved.     -   When cooled the solution is transferred to a 250 mL volumetric         flask, adjusted with deionised water and mixed.         1.7 Ammonium vanadate solution 0.24% 250 mL

(For Colour/Stop reagent)

Dissolve 0.5875 g ammonium Vanadate in 100 mL water preheated to 60° C.). Add slowly 5 mL 24.5% Nitric acid solution under continuous stirring. When cooled the solution is transferred to a 250 mL volumetric flask, adjusted with deionised water and mixed.

1.8 Colour/Stop reagent 200 mL

Should be prepared immediately before use.

Dispense 50 mL Ammonium heptamolybdate solution (10%) in a 200 mL volumetric flask followed by 50 mL of Ammonium Vanadate solution (0.24%). Dispense 33 mL 65% Nitric acid under continuous stirring. Adjust to 200.0 mL by addition of deionised water and mix.

Reaction Conditions

pH = 5.5 Incubation temperature = 37° C. ± 0.1° C. Incubation time = 60 minutes Preparation of a Phosphate Standard Curve (Absolute Method)

A standard curve using the KH₂PO₄ buffer (1.2) is prepared. The standard is diluted with Acetate buffer.

C_(dilution)=0.0 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 4.0 mM

Use the following equation: C _(stock solution) *V _(stock solution) =C _(dilution) *V _(dilution) V _(stock solution)=(C _(dilution) *V _(dilution))/C _(stock solution) Where C=concentration and V=volume

volume KH₂PO₄ Final Phosphate (18 mM) into 50 mL concentration volumetric Volume of in mM flask Acetate buffer 0 0.0 Ml 50.0 mL 0.5 1.4 mL 48.6 mL 1.0 2.8 mL 47.2 mL 1.5 4.2 mL 45.8 mL 2.0 5.6 mL 44.4 mL 2.5 6.9 mL 43.1 mL 3.0 8.3 mL 41.7 mL 4.0 11.1 mL  38.9 mL 1.00 mL of standard curve sample is mixed with 2.00 mL Colour/Stop reagent followed by 2.00 mL Phytate solution. The samples are incubated at room temperature for 5-10 minutes, and OD measured (as done with the samples after incubation). Sample Preparation:

1A: Liquid products (app. 5500 FTU/g): 1 g of sample is weighed in a 100 ml volumetric flask, the weight is written down, and the volumetric flask is filled up with Acetate buffer.

1B: Dry products (phytase formulated on wheat/starch carrier only): 1 g of product is weighed into a glass beaker. 100 mL of Acetate buffer is added by using a dispenser or measuring glass. The sample is stirred on a magnetic stirrer for 20 minutes. The extract is filtered through a glass filter.

1C: Dry products (coated granules with a hydrophobic outerlayer): Dry products: 1 g of product is weighed into a glass beaker. 100 mL of Acetate buffer is added by using a dispenser or measuring glass. The sample is stirred on a magnetic stirrer for 20 minutes. The extract is left for settling, coated product must not be filtered through a glass filter.

2: Prepare the further dilution of the sample. Preferred OD range is 0.4-1.6. If the concentration of the sample is not known, typical dilutions levels to bring the activity into the range for the assay are shown below.

The activity in the samples should be approximately 0.0300 U/mL.

The dilutions should be prepared immediately before determination.

Determination of Activity:

All samples are analysed at the same time—controls, blanks and samples. The assay run must be in a continuous flow.

All determinations are made as double or triple determinations.

A blank is included in the beginning of each series.

A control is included in each series of tubes (Phytase control or Phyzyme XP sample of known activity).

Blind samples: 1.00 mL of each sample is mixed with 2.00 mL Colour/Stop reagent followed by 2.00 mL Phytate solution. The samples are incubated at room temperature for 60 minutes, centrifuged and measured with the rest of the samples.

Samples:

1. Draw 1.00 mL of diluted sample or control 2 or 3 times in plastic tubes. For blanks draw 1.00 mL of Acetate buffer.

2. Place the Phytate solution (substrate) in a water bath at 37.0° C. for a minimum of 5 minutes until equilibrated to 37° C. 3.

3. Incubate the samples in the water bath at 37.0° C. for exactly 5 minutes.

4. Add 2.00 mL of equilibrated Phytate solution to the samples with a time interval of exactly 5 seconds.

5. Incubate in the water bath at 37.0° C. for exactly 60 minutes.

6. Add 2.00 mL Colour/Stop reagent, again with a time interval of 5 seconds, put on a lid and turn upside down 5 times.

7. Centrifuge for 10 min. at 3500 rpm.

8. Measure at 415 nm. Adjust the spectrophotometer to zero with demineralized water.

Calculation of Activity

OD of the blank has to be ≦0.13; if not the analysis has to be repeated.

Write the data in an Excel sheet

Make a standard curve and a tendency line in Excel:

a) y-axis=OD₄₁₅

b) x-axis=mM phosphate

c) the slope (α) has to be within 0.36-0.38

d) the intercept (β) must not exceed |0.02|

e) the correlation coefficient (R²) should be ≧0.99

The phytase activity is calculated as follows:

${{Activity}\mspace{14mu}\left( {{FTU}\text{/}g} \right)} = \frac{\left( {\left( {{OD}_{sample} - {OD}_{blank}} \right) - \beta} \right)*{DF}}{{Incubation}\mspace{14mu}{time}*\alpha*W_{sample}}$ OD_(sample)=mean absorbance of enzyme sample OD_(blank)=mean absorbance of enzyme blank DF=dilution factor for the sample Incubation time=60 minutes W_(sample)=weight of sample or control α=Slope of the standard curve β=Intercept of standard curve Phytase Assay at pH 3.5:

This is identical to the phytase assay at pH 5.5 with the following substitutions:

1.1 Assay buffer

Glycine-HCL buffer pH 3.5 (used instead of 1.1 Acetate buffer pH 5.5):

250 ml 0.2M glycin is mixed with 130 mL 0.2M HCl and app. 500 mL deionised water. pH is adjusted to 3.5 with 0.5 M HCl or 0.5 M NaOH. Transfer the solution to a 1000 mL volumetric flask and adjust the solution to 1000 mL by addition of deionised water

1.3 Phytate solution (substrate) 7.5 mM (in total 250 ml):

Dissolve 2.10 g (±0.05 g) Sodium phytate, deca hydrate (Phytic acid) in 200 mL assay buffer (Glycin HCl buffer pH 3.5).

Thermostate the solution to 37° C. in a water bath.

At 37° C. pH is adjusted to pH 3.5 by addition of 4 M HCl or 4 M NaOH.

Adjust to 250.0 mL by addition of assay buffer (pH 3.5). The bottle containing the solution needs to be wrapped in foil.

Shelf life: two weeks

A substrate correction factor may be needed when changing to another lot of phytic acid.

Correction factor for phytic acid batch 128H1234 (Sigma P-3168) is defined to 1,00

All dilutions of enzymes and phosphate in the method are done with Glycine-HCl assay buffer pH 3.5 instead of Acetate buffer pH 5.5

EXAMPLES Example 1 Introduction

To be efficient as an enzyme additive to food or animal feed, the phytase has to combine a number of different properties. In order to be able to degrade phytic acid in the acidic environment of an animal's stomach it has to be active at low pH, preferably over a broad range of pH values. In addition, it has to have high specific activity and preferably high thermostability to enable the protein to withstand high temperatures commonly used in preparation of feedstuffs such as feed pellets.

It is also important that the enzyme has broad substrate specificity allowing it to hydrolyse not only phytate but also intermediate products of phytate degradation such as inositol pentaphosphates, tetraphosphates and triphosphates. Studies on phytate degradation in pigs show that these inositol oligophosphates otherwise remain largely insoluble in the small and large intestine and thus inaccessible to alkaline phosphatases produced by the animal and gut microflora. Variations in substrate specificity profiles of different enzymes have been identified. For example, inositol-triphosphates generated by the phytase from B. subtilis are essentially resistant to further hydrolysis by this enzyme.

Suitably these variants show improved characteristics with respect to any one of the following: temperature stability, pH range, pepsin stability, specific activity, substrate specificity, and broader substrate specificity. Suitable methods for determining these characteristics are disclosed herein.

In particular, the improvements in phytase characteristics are directed to the enzyme stability under food and feed processing conditions, to the enzyme stability during stomach transit, and to the enzyme activity and stability in human or animal stomach and/or intestinal tract making the improved variants particularly suitable for use as feed supplements. Thus, such improvements comprise among other parameters the increase in stability at elevated temperatures, preferably at temperatures above 65° C., the increase in stability against proteolytic digestion, preferably protease of the digestive tract such as pepsin, the increase in catalytic activity at low pH, preferably catalytic activity below pH 5.5, and the general efficiency of releasing phosphate groups from phytate, and preferably in addition inositol phosphates.

Improvements in phytase characteristics are directed to the use in food and feed processing as well as for the use as an additive to food and feed products. In particular, improvements are directed to the stability under food and feed processing conditions, to the stability during stomach transit, and to the activity and stability in human or animal stomach and/or intestinal tract. Such improvements comprise among other parameters the increase in stability at elevated temperatures, preferably at temperatures above 65° C., the increase in stability against proteolytic digestion, preferably protease of the digestive tract, the increase in catalytic activity at low pH, preferably catalytic activity below pH 5.5, and the general efficiency of releasing phosphate groups from phytate.

The increase in stability at elevated temperatures is quantified by the inactivation temperature of the enzyme. The inactivation temperature is defined as the temperature at which the residual activity of a phytase enzyme after incubation for a certain duration and subsequent cooling to room temperature is 50% of the residual activity of the same phytase enzyme incubated for the same duration under the same conditions at room temperature. Thermostability differences are the differences in ° C. between the inactivation temperatures of two enzymes.

Example 2 Pepsin Stability

Pepsin resistance at pH 2 of phytases from Buttiauxella, variants BP-17, BP-110, BP-111, BP-112, and Phyzyme XP compared to Natuphos (BASF), and Ronozyme P (Novozymes/DSM).

Materials and Methods

Buffers:

Pepsin incubation buffer: 0.1 M Glycine-HCl, pH 2.0, 3 mg/ml BSA, 2.9 mg Sodium chloride anhydrous/mL, 0.73 mg calcium chloride/mL. For solutions with pepsin, the incubation buffer is prepared to contain 500, 1000, 3000, 6000, or 10000 U/ml of pepsin (Sigma P-7000, 10000 U/mg corresponds to 27 mg/ml), respectively. One pepsin unit is defined as the amount of enzyme that will produce a ΔOD₂₈₀ of 0.001 per min at pH 2.0 at 37° C., measured as TCA-soluble products using haemoglobin as substrate (Food Chemical Codex).

Phytase assay buffer: Acetate buffer 250 mM, pH 5.5

Phytase assay buffer with BSA: Acetate buffer 250 mM, pH 5.5, with 3 mg/ml BSA

Resistance Against Increasing Pepsin Concentration:

The set-ups for an enzymes were the same: Six samples with enzyme were prepared (in duplicate): Four samples with increasing amount of pepsin in buffer (pH 2), one sample without pepsin but in incubation buffer (pH 2), and one positive control sample with enzyme in assay buffer with BSA (pH 5.5).

For each sample, 900 μl incubation buffer without or with increasing amounts of pepsin or 900 μl assay buffer were mixed with 100 μl enzyme solution followed by incubation at 40° C. After 120 min incubation, 100 μl was withdrawn and mixed with 900 μl assay buffer. Samples were immediately analysed for phytase at pH 5.5 against phosphate standard curve as described by ISO draft international standard ISO/DIS 30024.

Results

Pepsin Resistance

The Buttiauxella variants an showed excellent stability towards pepsin at pH 2. In contrast, the activity of Natuphos was reduced dramatically already at a pepsin concentration of 500 U/ml, with further reduced activity finding a plateau of about 45% recovery at a pepsin concentration 3000 U/ml. The recovery of Ronozyme P was even worse with a reduction in recovery of less than 20% at a pepsin concentration of only 500 U/mg (FIG. 9A).

FIG. 9 shows the resistance of the phytases originating from Buttiauxella, variants BP-17, BP-110, BP-111, and BP-112, and of Phyzyme XP, Natuphos, and Ronozyme P against increasing concentrations of pepsin. Data are relative to incubation at pH 2 without pepsin. A: all data points, B: same data but showing only more than 70% recovery.

TABLE 1 Same data as presented in FIG. 9 Pepsin level BP-17 BP-17 v. 110 BP-17 v. 111 BP-17 v. 112 Phyzyme XP Natuphos Ronozyme P, new 0 100% 100% 100% 100% 100% 100% 100% 500 87% 96% 98% 85% 61% 19% 1000 81% 98% 96% 85% 51% 16% 3000 82% 96% 94% 84% 93% 44% 17% 6000 82% 92% 89% 80% 89% 45% 18% 10000 86% 95% 94% 79% 98% 46% 21%

There are slight differences between the Buttiauxella variants, where variant BP-17 var. 110 shows best stability having 95% activity left after two hours of incubation at the highest pepsin concentration, 10000 U/mL (FIG. 9B). In comparison, BP-17 showed a recovery of around 85% after two hours of incubation.

The Buttiauxella phytases also showed excellent acid stability. Comparing the activity measured at pH 5.5 after two hours of incubation at either pH 2 or pH 5.5 showed that all four Buttiauxella phytases did not lose activity during the 2 hours incubation at pH 2 whereas Phyzyme XP, Natuphos, and the new Ronozyme P showed slight to significant reduction in activity (Table 2).

TABLE 2 The activity measured at pH 5.5 after incubation in a buffer at pH 2 or pH 5.5 for two hours. The numbers are relative to the activity from the samples incubated at pH 5.5. BP-17 BP-17 v. 110 BP-17 v. 111 BP-17 v. 112 Phyzyme XP Natuphos Ronozyme P, new Assay buffer 100% 100% 100% 100% 100% 100% 100% pH 2, no pepsin 101% 102% 106% 120% 83% 90% 67% Data Summary

The Buttiauxella phytase variants according to the present invention show more than 75% recovery after 2 hours of incubation at pH 2, 37° C., 10000 U/ml of pepsin compared to activity of the sample incubated under the same condition but without pepsin present.

Example 3 Recovery of Enzyme Activity

Evaluation of Recovery of Enzyme Activity

Materials and Methods

Here, we evaluate the recovery of enzyme activity after pelleting of the wheat formulated enzyme. The enzyme is sprayed on granulated wheat and dried prior to mixing with feed followed by the pelleting process.

Liquid enzymes where formulated on whole grounded wheat and dried to a dry matter content on approximately 90%. The inactivation of enzymes during thermal stress in the pelleting process was tested using a pelleting unit at the Technological Institute in Kolding (schematically presented in FIG. 10). Test sample is added to 10 kg premix and mixed for 10 min. Then 10 kg premix was added to 150 kg feed (large horizontal mixer) and mixed for 15 min. before conditioning/steaming. Feed is treated 30 sec. at 90° C./95° C. before pelleting. The temperature is measured in the outlet of the cascade mixer. Immediately after leaving the pelleting press the pellets are cooled to room temperature.

Phytase activity was measured according to the methodology recited herein.

Phytase activity is 5 units per gm in the mash feed prior to pelletising.

Results

The results are presented in FIGS. 11 to 13.

In FIG. 11, the results from the pelleting trials of three Phytase B variants according to the present invention is disclosed. The enzymes were formulated on whole grounded wheat and dried to app 10% water content. Here, the recovery of phytase activity after pelleting for BP 17 var 111 is 90% at 90° C. compared to a recovery of 19% at 90° C. for the BP 17.

As reference the Phyzyme XP, an E. coli phytase was formulated on whole grounded wheat. The results are shown in FIG. 12. Here, the recovery of phytase activity after being pelletised at 90° C. from the three batches of Phyzyme XP formulated on whole grounded wheat is 47%.

Ronozyme was also tested. The product is sold in a coated version, to protect the enzyme from the heat in the pelleting process. In this trial the phytase was extracted from the coated product and formulated on whole grounded wheat to study the themostability of the phytase molecule without protection. The results are shown in FIG. 13.

Data Summary

The Buttiauxella phytase variants of the present invention formulated on wheat show more than 70% recovery after being pelleted at 90° C.

Example 4 Phytic Acid Degradation

Results from a Study of Phytic Acid Degradation by Phytase

Solutions

Three different buffers were used in the incubations. They were as follows:

250 mM acetate pH 5.5;

250 mM Acetate M pH 3.5; and

250 mM HCl glycine pH 2.5.

All phytases used on the incubations were diluted in assay buffer to enzyme level at 1 U/ml.

The phytate substrate solution used in the incubation is a Phytate 1% (1 g/100 ml buffer).

The substrate is prepared in same buffer as used for the dilution of phytases to keep the pH level constant in the reaction.

The reaction is terminated by 1M HCl.

Incubation

The incubation volumes are: 1.5 or 3.0 ml Phytate+0.25 ml enzyme+3.25 or 1.75 ml buffer at 37° C., a total volume of 5.0 ml.

Sub-samples of 0.5 ml are taken at different time points (0, 30, 60 and 90 min).

An eppendorf tube is prepared with a 0.2 ml 1M HCl for each sub-sample prior to the sub-sampling. By blending the sub-sample from the incubation with HCl the enzyme reaction is terminated. Each sub-sample of 0.7 ml is stored at 4° C. until HPLC analysis.

HPLC Method

Analysis of phytate and inositol isomers by high performance ion chromatography (HPLC). This method has been described by Skoglund and Carlsson et al. (J. Agric. Food Chem., 45 (1997), 451-436 5. J. Agric. Food Chem., 46 (1998), 1877-1882; and J. Agric. Food Chem., 49 (2001), 11695-1701. The column used was a strong anion exchanger (4×250 mm) from Dionex with a pre-column of 4×50 mm. Solvent A, MilliQ water, Solvent B, 1N HCl prepared in water. The gradient is from 2.5% to 49% B in 30 min followed by 3 min of isocratic at 50% and 2 min isocratic at 2.5% at a flow of 0.8 ml/min. Each run is 35 min. It is possible to reduce the run to totally 25 min as phytase does not produce so many theoretically possible IP isomers. The eluent was in-line derivatized with a water solution containing 0.1% Fe(NO₃)₃.9HO₂ and 2% perchloric acid (HClO₄) at a flow of 0.4 ml/min. Phytate and IP isomers were detected at 290 nm as a positive peak. This is due to the formation of phytate-Fe³⁺—ClO₄ ⁻ complex. A perchloric acid solution of 60% was bought from Sigma.

Results

The results are presented in FIGS. 14-17.

Data Summary

All of the enzymes of the present invention display favourable characteristics.

The phytases of the present invention break down phytate at all of the tested pH levels.

The enzymes of the present invention are very active at pH 5.5.

BP 110 and BP 111 display similar activity at pH 2.5 and 3.5.

BP 110 and BP 111 break down all the phytate added to the incubations after 90 min at both pH 2.5 and pH 3.5

BP112 breaks down 75% of the phytate added to the incubations after 90 min at both pH 2.5 and pH 3.5

The enzymes of the present invention display better characteristics than Ronozyme and Natuphos at pH 2.5 (see at least FIG. 17) (Enzyme concentration is higher than seen in FIGS. 14-16, so Buttiauxella BP17 is there as reference.)

Example 5 Phytic Acid Hydrolysis in a Liquefact

Phytic Acid Determination:

Phytic acid content: Phytic acid was extracted from a sample by adjusting the pH of the 5% slurry (if it is dry sample) to pH 10 and then determined by an HPLC method using an ion exchange column. Phytic acid was eluted from the column using a NaOH gradient system. Phytic acid content in the liquid was then calculated by comparing to a phytic acid standard.

Results

The effect of temperature on the hydrolysis of phytic acid of the whole ground corn liquefact from a conventional dry grind liquefaction process (source: Illinois River Energy, Monroe, Ill.) by different thermostable BP variant phytase, i.e., BP110, BP111 and BP112 was studied. The pH of a 32% ds (“dry solid”) whole ground corn ds corn liquefact was adjusted to pH 5.0 and placed in a water bath maintained at 85° C. and 89° C. After temperature equilibration, BP-phytase was added at 4.0 FTU/gds. corn. Samples were then taken at 20 minutes and the enzyme reaction was terminated by the addition of 10 mM sodium hydroxide (diluted 1 to 10 fold). The diluted samples were then filtered and analyzed by HPLC for their phytate derivatives profile (IP1 to IP6). The HPLC chromatograms in FIGS. 18 and 19 clearly showed that phytase from all three variants catalyzed the hydrolysis of phytic acid at temperature greater than 85° C. The phytic acid content (phytic acid (IP6) and intermediates IP1 to IP5) in whole ground corn liquefact is around 1.7% ds.corn and data in FIG. 18 showed that more than 95% of the phytic acid was hydrolyzed by thermostable phytase under the current liquefaction conditions. Significantly, the HPLC profile from the samples incubated at 89° C. showed that the BP-111 phytase variant exhibited higher thermostability compared to phytase from two other variants (see FIG. 19; BP-110 and BP-112).

Example 6 Expression of the Aspergillus tubingensis Lipase 3 in Trichoderma reesei

1. Expression Construct & Strain Used for Transformation

pDONR™221 plasmid DNA obtained from Invitrogen (catalogue no. 12536-017) was used as the donor vector. pDONR221::lip 3 containing the Aspergillus tubingensis lipase 3 genomic DNA (FIG. 20) was recombined into the T. reesei gateway destination vector pTrex3G (described in detail in WO 05/001036), resulting in the final expression construct ATlipase3Trex (FIG. 21).

The expression cassette contained the promoter and terminator regions of the T. reesei cellobiohydrolase 1 (cbh1) gene. It also contained the Aspergillus nidulans acetamidase, amdS gene as a selectable marker for transformation of T. reesei.

The Aspergillus tubingensis lipase 3 genomic DNA encodes a lipase 3 lipolytic enzyme having the amino acid sequence shown in SEQ ID No. 7.

The term “lipase 3” when used herein refers to a lipolytic enzyme comprising the amino acid sequence shown in SEQ ID No. 8, such as the amino acid sequence shown in SEQ ID No. 7. Seq. ID No. 7 contains the signal seq and seq. ID No. 8 is the mature lipase protein without the signal sequence.

The strain used for transformation was Trichoderma reesei, a derivative of the non-GMM strain RL-P37 from which the genes encoding the two secreted cellobiohydrolases, CBHI and CBHII, and two of the secreted endoglucanases, EGI and EGII, have been deleted.

The Expression Vector pTrex3g.

The following describes the construction of the vector pTrex3g which may be used to express the genes of the present invention.

This vector is based on the E. coli vector pSL1180 (Pharmacia Inc., Piscataway, N.J., USA) which is a pUC118 phagemid based vector (Brosius, J. (1989) DNA 8:759) with an extended multiple cloning site containing 64 hexamer restriction enzyme recognition sequences. It was designed as a Gateway destination vector (Hartley, J. L., Temple, G. F. and Brasch, M. A. (2000) Genome Research 10:1788-1795) to allow insertion using Gateway technology (Invitrogen) of any desired open reading frame between the promoter and terminator regions of the T. reesei cbhl gene. It also contains the Aspergillus nidulans amdS gene for use as a selectable marker in transformation of T. reesei.

The details of pTrex3g are as follows. The vector is 10.3 kb in size.

Inserted into the polylinker region of pSL1180 are the following segments of DNA:

-   -   1. A 2.2 bp segment of DNA from the promoter region of the T.         reesei cbhl gene     -   2. The 1.7 kb Gateway reading frame A cassette acquired from         Invitrogen that includes the attR1 and attR2 recombination sites         at either end flanking the chloramphenicol resistance gene (CmR)         and the ccdB gene     -   3. A 336 bp segment of DNA from the terminator region of the T.         reesei cbh1 gene     -   4. A 2.7 kb fragment of DNA containing the Aspergillus nidulans         amdS gene with its native promoter and terminator regions.         2. Transformation of T. reesei Quad Delete Host Strain

The expression construct, ATlipase3Trex, containing the A. tubingensis lipase 3 gene was transformed into a T. reesei strain using electroporation or biolistic transformation by particle bombardment using the PDS-1000 Helium system (BioRad Cat. No. 165-02257). PCR products containing only fungal DNA or the entire expression plasmid were used for generating transformants by biolistic transformation and electroporation.

A. Transformation by Electroporation

The T. reesei host strain to be transformed was grown to full sporulation on PDA plates for 5 days. Spores from 2 plates were harvested with 1.2M sorbitol and filtered through miracloth to get rid of the agar. Spores were transferred to a 50 ml falcon tube and washed by repeated centrifugation 5-6 times with 50 ml water. The spores were resuspended in a small volume (less than 2× pellet volume) using 1.2M sorbitol solution. The spore suspension was then kept on ice. 90 ul of spore suspension was aliqouted into the electroporation cuvette (E-shot, 0.1 cm standard electroporation cuvette from Invitrogen). 10-20 ul DNA construct (plasmid FIG. 4 or PCR product) were added to the spore suspension and electroporation was set at 16 kV/cm, 24 μF, 50Ω. After electroporation, the spore suspension was left on ice, resuspended in 5 parts 1.0M sorbitol and 1 part YEPD, and allowed to germinate by incubation at 28 C with shaking 250 rpm overnight. The next day, the germlings were plated in agar plates containing acetamide. Transformants were picked and transferred individually to acetamide agar plates.

B. Transformation by Particle Bombardment (Biolistic Transformation)

A suspension of spores from a quad deleted strain of T. reesei was prepared. 200 ul of spore suspension was spread onto the center of the minimal medium (MM) acetamide plates. (MM acetamide plates had the following composition: 0.6 g/l acetamide; 1.68 g/l CsCl; 20 g/l glucose; 20 g/l KH2PO4, 0.6 g/l CaCl2 2H20; 1 ml/l 1000× trace elements solution; 20 g/l Noble agar, and pH5.5. 1000× trace elements solution contained 5.0 g/l FeSO4 7H2O; 1.6 g/l MnSO4; 1.4 g/l ZnSO4 7H2O and 1.0 g/l CoCl2 6H2O. The spore suspension was allowed to dry on the surface of MM acetamide medium for 1 hour in the sterile hood. Transformation followed the manufacturer's instruction. 60 mg of tungsten particles were placed in a microfuge tube. 1 ml of ethanol was added and allowed to stand for 15 seconds. The ethanol was removed and the particles were washed three times with sterile dH₂O before 250 ul of 50% (v/v) sterile glycerol was added. 25 ul of tungsten particle suspension was placed onto a microfuge tube. While continuously vortexing, the following were added: 5 ul (100-200 ng/ul) of plasmid DNA, 25 ul of 2.5M CaCl₂ and 10 ul of 0.1M spermidine. The particles were centrifuged for 3 seconds. The supernatant was removed and the particles were washed with 200 ul of 100% ethanol and centrifuged for 3 seconds. The supernatant was removed. 24 ul

100% ethanol was added and mixed by pipetting, then 8 ul aliquots of particles were removed and placed in the centre of microcarrier disks that were held in a desiccator. Once the tungsten/DNA solution had dried the microcarrier disk was placed in the bombardment chamber along with the plate of MM acetamide with spores and the bombardment process was carried out according to the manufacturer's instructions. After bombardment of the plated spores with the tungsten DNA particles, the plates were incubated at 28° C. Transformed colonies were transferred to fresh plates of MM acetamide medium and incubated at 28° C.

3. Growth of Transformants in Microtiter Plates

After 5 days of growth on MM acetamide plates, transformants obtained by electroporation or by biolistic transformation and displaying stable morphology were inoculated into 200 ul Defined medium with glucose/sophorose in a 96-well microtiter plates. Defined medium with glucose/sophorose (per liter) consists of NH₄2SO₄ 5 g, PIPPS buffer 33 g, Casamino Acids 9 g, KH₂PO₄ 4.5 g, CaCl₂ (Anhydrous) 1 g, MgSO₄.7H₂O 1 g, pH to 5.50 adjusted with 50% NaOH with milli-Q H2O bring to 966.5 mL. After sterilization, the following were added: Mazu 5 mL, Glucose/Sophrose 60% 26 mL and 400×T. reesei Trace Metals 2.5 mL. The microtiter plate was incubated in an oxygen growth chamber at 28° C. for 5 days.

Example 7

The expression host cells suitable for use in the present invention may be a strain of T. reesei in which the genes encoding cellobiohydrolase I (CBHI, Cel7a), cellobiohydrolase II (CBHII, Cel6a), endoglucanase I (EGI, Cel7b), and endoglucanase II (EGII, Cel5a) have been inactivated by deletion or disruption using molecular genetic techniques. This strain (a quad delete strain) is useful as a host for over-expression of genes encoding other T. reesei secreted proteins.

Preferably host cells suitable for use in the present invention may be derived from a Trichoderma reesei cell of the strain RL-P37 using the methods described in WO 2005/001036 and US28026376 A1.

A T. reesei strain suitable for use in the present invention may be derived from the publicly available strain of T. reesei RL-P37. T. reesei strain RL-P37 may be modified to form T. reesei strain 1A52 as described in WO 05/001036. T. reesei strain 1A52 may be modified as described in US 20080026376 to form a T. reesei cell usable in the present invention.

RL-P37 may be modified to form T. reesei strain 1A52 as described below.

The T. reesei host strain used may be derived from the publicly available strain RL-P37 which has previously been used to manufacture commercial cellulase preparations by Genencor International, Inc. The derivation and characterisation of this strain has been published previously (Sheir-Neiss, G. and Montenecourt, B. S. (1984) Appl. Microbiol. Biotechnol. 20:46-53; U.S. Pat. No. 4,797,361). It is a cellulase over-producing mutant strain which has been obtained as a result of several mutagenesis steps from the wild-type strain (QM6a).

1) Isolation of a pyr4 Mutant Strain.

In order to prepare strain RL-P37 for transformation with plasmid DNA it was necessary to isolate a derivative having a null mutation in the pyr4 gene.

The pyr4 gene encodes orotidine-5′-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. The toxic inhibitor 5-fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells. However, cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for pyr4 mutant strains using FOA. In practice, spores of T. reesei strain RL-P37 were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resistant colonies appeared within three to four days. FOA-resistant mutants which required uridine for growth were identified. In order to identify those mutants which specifically had a defective pyr4 gene protoplasts were generated and transformed with a plasmid containing a wild-type pyr4 gene (Smith, J. L., Bayliss, F. T. and Ward, M. (1991) Curr. Genet. 19:27-33). Following transformation protoplasts were plated on medium lacking uridine. Subsequent growth of transformed colonies demonstrated complementation of a defective pyr4 gene by the plasmid-borne pyr4 gene. In this way strain GC69 was identified as a pyr4 mutant of strain RL-P37.

2) Construction of a Plasmid Designed to Delete the CBH1 Encoding Gene.

The cbhl gene, encoding the CBHI protein, was cloned from the genomic DNA of strain RL-P37 by hybridization with an oligonucleotide probe designed on the basis of the published sequence for this gene (Shoemaker, S., Schweickart, V., Ladner, M., Gelfand, D., Kwok, S., Myambo, K. and Innis, M. (1983) Biotechnology 1:691-696). The cbhl gene resides on a 6.5 kb PstI fragment and was inserted into the PstI site of pUC4K (Pharmacia Inc., Piscataway, N.J., USA) replacing the kanamycin-resistance gene of this vector. The resulting plasmid, pUC4K::cbh1, was then cut with Hindlll and the larger fragment was isolated and religated to give pUC4K::cbh1ΔH/H. This procedure removed the entire cbhl coding sequence and approximately 1.2 kb of 5′ and 1.5 kb of 3′ flanking sequences. Approximately 1 kb of flanking DNA remained from either end of the original PstI fragment. The T. reesei pyr4 gene was cloned as a 6.5 kb Hindlll fragment of genomic DNA in pUC18 to form pTpyr2 (Smith, J. L., Bayliss, F. T. and Ward, M. (1991) Curr. Genet. 19:27-33). The plasmid pUC4K::cbh1ΔH/H was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This DNA was ligated with the 6.5 kb Hindlll fragment containing the pyr4 gene to give pΔCBHlpyr4.

Digestion of pΔCBHlpyr4 with EcoRl liberated a larger fragment which consisted of flanking regions of the cbhl locus at either end with the pyr4 gene replacing the cbhl coding sequence in the centre. The only DNA on this fragment which was not derived from T. reesei was a 21 bp fragment derived from the multiple cloning site of pUC4K.

3) Deletion of the cbhl Gene of T. reesei.

Protoplasts isolated from mycelium of strain GC69 were transformed with EcoRl digested plasmid pΔCBHlpyr4 using methods outlined by Smith et al., 1991. Stable transformants were obtained and those from which the cbhl gene had been deleted were identified as described below.

Total DNA was isolated from the transformants, digested with PstI, subjected to agarose gel electrophoresis and blotted to a membrane filter. The filter was then hybridised with P³² labelled pΔCBHlpyr4 and the pattern of hybridisation observed by autoradiography. This probe hybridised with the native cbhl and pyr4 genes in an untransformed strain. In one transformant (strain P37PΔCBHI) a pattern of hybridisation was observed which would be predicted if a double cross-over integration event had occurred. That is, the cbhl gene had been deleted by integration of a single copy of the larger EcoRl fragment obtained from pΔCBHlpyr4 at the cbhl locus of strain RL-P37.

Southern analysis was also performed as above except that the probe used was radiolabelled pintCBHI. This plasmid consists of a pUC vector containing a 2 kb Bglll fragment from the cbhl locus within the region that was deleted in pUC4K::cbh1ΔH/H. This plasmid hybridised to the cbhl locus of strain GC69 but did not hybridise to DNA from strain P37PΔCBHI. This confirms that the cbhl gene had been deleted and that the pUC DNA fragment from pΔCBHlpyr4 had not been incorporated by the deleted strain.

Analysis of secreted proteins by separation on isoelectric focusing gels showed that the CBH1 protein was not produced by strain P37PΔCBHI.

4) Generation of a pyr4 Null Mutant of P37PΔCBHI.

Spores of the transformant (P37PΔCBHI) which was deleted for the cbhl gene were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section above. This pyr4 deficient strain was designated P37PΔCBHIPyr⁻26. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PΔCBHIPyr⁻26 was selected. This deletion completely removed the pyr4 gene which had integrated at the cbhl locus in strain P37PΔCBHI, as well as flanking DNA from the cbhl locus beyond the extent of the 6.5 kb PstI fragment of genomic DNA which was originally cloned.

5) Construction of a Vector Designed to Delete the cbh2 Gene.

The cbh2 gene of T. reesei, encoding the CBHll protein, has been cloned as a 4.1 kb EcoRl fragment of genomic DNA (Chen et al., 1987, Biotechnology 5:274-278). This 4.1 kb fragment was inserted between the EcoRl sites of pUC4XL. The latter plasmid is a pUC derivative (constructed by R. M. Berka, Genencor International Inc.) which contains a multiple cloning site with a symetrical pattern of restriction endonuclease sites arranged in the order shown here. EcoRI, BamHI, Sacl, Smal, Hindlll, Xhol, Bglll, Clal, Bglll, Xhol, Hindll I, Smal, Sacl, BamHI, EcoRI. The plasmid, pPΔCBHll was constructed in which a 1.7 kb central region of this cbh2 clone, between a HindIII site (at 74 bp 3′ of the CBHll translation initiation site) and a ClaI site (at 265 bp 3′ of the last codon of CBHII), has been removed and replaced by a 1.6 kb HindIII-Clal DNA fragment containing the T. reesei pyr4 gene obtained as follows. The T. reesei pyr4 gene was excised from pTpyr2 on a 1.6 kb Nhel-Sphl fragment and inserted between the Sphl and Xbal sites of pUC219 (derived from pUC119 by expanding the multiple cloning site to include restriction sites for Bglll, Clal and Xhol; Wilson et al., 1989, Gene 77:69 78) to create p219M (Smith et al., 1991, Curr. Genet. 19:27-33). The pyr4 gene could then be removed as a HindIII-Clal fragment having seven by of DNA at one end and six by of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pPΔCBHII.

Digestion of this plasmid with EcoRl liberated a fragment having 0.7 kb of flanking DNA from the cbh2 locus at one end, 1.7 kb of flanking DNA from the cbh2 locus at the other end and the T. reesei pyr4 gene in the middle. The only DNA in this fragment which was not derived from T. reesei was the 6 bp and 7 bp fragments of the pUC219 multiple cloning site at either end of the pyr4 gene.

6) Deletion of cbh2 Gene from Strain P37PΔCBHIPyr⁻26

Protoplasts of strain P37PΔCBHIPyr⁻26 were generated and transformed with EcoRl digested pPΔCBHII according to the methods outlined in 3 above. Stable transformants were cultured in shake flasks and the protein in the culture supernatants was examined by isoelectric focussing. One transformant (designated P37PΔΔCBH67) was identified which did not produce any CBHll (nor CBHI) protein.

DNA was extracted from strain P37PΔCBH67, digested with EcoRl and Asp718, and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with ³²P labelled pPΔCBHII. The 4.1 kb EcoRl fragment containing the wildtype cbh2 gene was observed in the DNA from an untransformed control strain. In contrast, in strain P37PΔCBH67 the single 4.1 kb band was eliminated and replaced by two bands of approximately 0.9 and 3.1 kb. This is the expected pattern if a single copy of the larger EcoRl fragment from pPΔCBHll had integrated precisely at the cbh2 locus and deleted the cbh2 gene.

The same DNA samples were also digested with EcoRl and Southern analysis was performed as above. In this example the probe was ³²P labelled plntCBHII. This plasmid contains a portion of the cbh2 gene coding sequence from within that segment of cbh2 DNA which was deleted in plasmid pPΔCBHII. No hybridization was seen with DNA from strain P37PΔCBH67 confirming that the cbh2 gene was deleted and that the pUC plasmid fragment of pPΔCBHlI had not been incorporated by this strain.

7) Selection of a pyr4 Null Mutant of Strain P37PΔΔCBH67.

Spores of the transformant (P37PΔΔCBH67) which was deleted for both the cbhl and cbh2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section 1 above. This pyr4 deficient strain was designated P37PΔΔCBH67Pyr⁻1. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PΔΔCBH67Pyr⁻1 was selected. This deletion completely removed the pyr4 gene which had integrated at the cbh2 locus in strain P37PΔCBH67, as well as flanking DNA from the cbh2 locus beyond the extent of the 4.1 kb EcoRl fragment of genomic DNA which was originally cloned. The short (6 bp and 7 bp) fragments of DNA derived from the pUC219 multiple cloning site which were present at either end of the pyr4 gene would also have been removed from the genome by this deletion.

8) Construction of a Plasmid Designed to Disrupt the egl2 Gene.

The egl2 gene, encoding EGll (previously referred to as EGlll by some), has been cloned from T. reesei and the DNA sequence published (Saloheimo et al., 1988, Gene 63:11-21). We have obtained the gene from strain RL-P37 as an approximately 4 kb Pstl-Xhol fragment of genomic DNA inserted between the Pstl and Xhol sites of pUC219. The T. reesei pyr4 gene, present on a 2.7 kb SalI fragment of genomic DNA obtained from pTpyr2, was inserted into a SalI site within the EGll coding sequence to create plasmid pEGII::P-1. This resulted in disruption of the EGll coding sequence but without deletion of any sequences. The plasmid, pEGII::P-1, can be digested with HindIII and BamHl to yield a linear fragment of DNA derived exclusively from T. reesei except for 5 bp on one end and 16 bp on the other end both of which are derived from the multiple cloning site of pUC219.

9) Disruption of the eg12 Gene of Strain P37PΔCBH67Pyr⁻1.

Strain P37PΔΔCBH67Pyr⁻1 was transformed with pEGII::P-1 which had been previously digested with Hindlll and BamHl and stable transformants were selected. Total DNA was isolated from transformants and Southern analysis used to identify strains in which the fragment of plasmid DNA containing the pyr4 and egl2 genes had integrated at the egl2 locus and consequently disrupted the EGll coding sequence. Southern analysis was performed using as a probe an approximately 4 kb Pstl fragment of T. reesei DNA containing the eg12 gene. When DNA isolated from strain P37PΔΔ67P⁻1 was digested with Pstl for Southern analysis the egl2 locus was subsequently visualised as a single 4 kb band on the autoradiograph. However, for a transformant disrupted for the egl2 gene this band was lost and was replaced by two new bands as expected. When the DNA was digested with Bglll or EcoRV the size of the band corresponding to the egl2 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37PΔΔ67P⁻1 strain and the transformant disrupted for egl2. This latter transformant, now deleted for the cbhl, cbh2, and egl2 genes, was designated as strain B31. Further Southern analysis confirmed that the pUC DNA fragment of pEGII::P-1 was not incorporated in this strain.

10) Selection of a pyr4 Null Mutant of Strain B31.

Spores of the transformant (B31) which was deleted for the cbhl, cbh2 and egl2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section 1 above. This pyr4 deficient strain was designated B31 P6. Southern analysis has shown that a spontaneous deletion had occurred when strain B31P6 was selected. This deletion removed the majority of the pyr4 gene which had integrated at the eg12 locus in strain B31, but did not extend into the flanking DNA of the eg12 locus.

11) Construction of a Plasmid Designed to Delete the egl1 Gene.

The egl1 gene of T. reesei has been cloned and the DNA sequence of the gene has been published (Penttila et al., 1986, Gene 45; 253-263; van Arsdell et al., 1987, Biotechnology 5:60-64). We have obtained this gene from T. reesei strain RL-P37 as a 4.2 kb HindIII fragment of genomic DNA inserted at the HindIII site of pUC100 (a derivative of pUC18 with an oligonucleotide inserted into the multiple cloning site adding restriction sites for Bglll, Clal and Xhol) to give pUCEGI. An approximately 1 kb EcoRV fragment extending from a position close to the middle of the EGI coding sequence to a position beyond the 3′ end of the coding sequence was removed and replaced by a 3.5 kb Scal fragment of T. reesei DNA containing the pyr4 gene obtained from pTpyr2. The resulting plasmid was called pPΔEGI.

The plasmid, pPΔEGl could be digested with HindIII to release a DNA fragment comprising only T. reesei genomic DNA having a segment of the egl1 gene at either end and the pyr4 gene, replacing part of the EGI coding sequence, in the centre.

12) Deletion of the eal1 Gene in Strain B31P6.

Two forms of pPΔEGl were constructed which differed only in the orientation of the pyr4 gene with respect to the egl1 flanking regions. Strain B31P6 was transformed with a mixture of both forms of the plasmid after they had been digested with Hindlll. Total DNA was extracted from stable transformants, digested with Hindlll and subjected to Southern analysis. The probe used was radio labelled pUCEGI. Hybridisation was observed to a 4.2 kb fragment of DNA from strain B31P6 representing the undeleted egl1 gene. A transformant (strain 1A52) was identified in which this 4.2 kb was no longer present but had been replaced by a fragment of approximately 6.8 kb. This is the pattern expected if the larger HindIII fragment from pPΔEGI had integrated precisely as predicted at the egl1 locus leading to deletion of part of the EGI coding sequence and insertion of pyr4 at this position. Using a pUC plasmid as a probe for Southern analysis it was confirmed that the pUC DNA fragment of pPΔEGI had not been incorporated in strain 1A52.

T. reesei strain 1A52 may be modified to form a T. reesei cell usable in the present invention.

Example 8 Nutrient Digestibility of Diets Supplemented with a Combination of Phytase and Lipase Together with Amylase and Xylanase in Cannulated Piglets

This trial was performed at the University of Illinois, USA.

Materials and Methods

The ileal energy and protein digestibility, total tract energy digestibility, and total tract Ca and P retention of corn/soybean meal based diets supplemented with lipase on top of a phytase, amylase and xylanase in combination in surgically cannulated piglets was evaluated. The treatments consisted of:

Positive control. Nutrient adequate diet.

Negative control. Reduced in 150 kcal of DE/kg of feed, 0.15% Calcium, and 0.12% available P compared to positive control.

Phytase (500 FTU/kg).

Phytase (500 FTU/kg), amylase (1800 u/kg) and xylanase (2000 u/kg).

Phytase (500 FTU/kg), amylase (1800 u/kg), xylanase (2000 u/kg) and lipase (3000 LIPU/kg).

Twelve barrows were randomly allotted to a duplicated 6×6 Latin square design with 6 diets and 6 periods per square each. Individual pigs were identified by ear tags.

Diet formulations and calculated composition for the positive control and the negative control are presented in Table 3. Diets were mixed by supplementing enzyme products to the negative control (NC) at the expense of cornstarch. The basal diet was based on corn, soybean meal, and corn DDGS. Chromic oxide at 0.4% was added to the diets as indigestible marker.

TABLE 3 Diet formulation and nutritional specifications PC NC (%) Ingredient Corn 51.05 57.05 Soybean meal 48% 24.00 21.75 DDGS 3.50 3.50 Whey, dried 12.00 12.00 Fish meal 4.00 4.00 Soy oil 3.00 0.00 Starch or enzyme 0.05 0.05 Dicalcium phosphate 0.85 0.10 Limestone 0.60 0.60 Salt 0.40 0.40 Lysine-HCl 0.20 0.20 DL-methionine 0.00 0.00 L-threonine 0.05 0.05 Premix¹ 0.30 0.30 Total 100.00 100.00 Calculated nutrient composition DE (kcal/kg) 3,570 3,420 NE (kcal/kg) 2,500 2,360 Starch 31.12 34.70 NSP 8.87 9.05 CP (%) 20.54 19.97 Crude fat (%) 6.16 3.40 Calcium (%) 0.81 0.64 Av. phosphorus (%) 0.40 0.28 Dig. Lysine (%) 1.19 1.14 Dig. Methionine (%) 0.32 0.32 Dig. TSAA (%) 0.66 0.65 Dig. Threonine (%) 0.74 0.71 ¹Provide the following quantities of vitamins per kilogram of complete diet: Vitamin A, 10,990 IU; vitamin D3, 1,648 IU; vitamin E, 55 IU; vitamin K, 4.4 mg; thiamin, 3.3 mg; riboflavin, 9.9 mg; pyridoxine, 3.3 mg; vitamin B12, 0.044 mg; D-pantothenic acid, 33 mg; niacin, 55 mg; folic acid, 1.1 mg; biotin, 0.17 mg; Cu, 16 mg as copper sulfate; Fe, 165 mg as iron sulfate; I, 0.36 mg as potassium iodate; Mn, 44 mg as manganese sulfate; Se, 0.3 mg as sodium selenite; and Zn, 165 mg as zinc oxide.

All pigs used in this study originated from the mating of line 337 boars and C22 females (Pig Improvement Company, Franklin, Ky.). Barrows with an average initial body weight of 12.5 kg (SD=2.15) were used. Pigs were surgically fitted with a T-cannula in the distal ileum. Following the surgery, pigs were housed in individual pens. Animals were fed conventional post-weaning diet until the initiation of the experiment. Feed was provided at daily levels of three times the estimated maintenance requirement for energy (i.e. 106 kcal ME/kg BW0.75). The feed allowance was adjusted at the beginning of each period when pig BWs were recorded.

The enzyme products were orally administered mixed with a diet for 7 days in each period: 4 days for adaptation, 1 day for fecal collection, and 2 days for ileal digesta collection. Ileal digesta samples were collected for 8 h on d 6 and 7. Briefly, a plastic bag was attached to the cannula barrel using a cable tie and digesta flowing into the bag were collected. Bags were removed whenever they were filled with digesta, or at least once every 30 minutes. Collected samples were stored at −20° C. to prevent bacterial degradation of amino acids in the digesta. At the conclusion of the experiment, frozen ileal samples were allowed to thaw at room temperature, mixed within animal and diet, and a sub-sample was collected for chemical analysis. Ileal digesta samples were lyophilized and finely ground prior to chemical analysis.

Ileal samples were analyzed for dry matter (procedure 930.15; AOAC, 2005). Gross energy concentration in ileal samples were measured using bomb calorimetry (Parr 6300 calorimeter, Parr Instruments Co., Moline, Ill.). The Cr contents of diets and ileal digesta samples were measured using an ICP method (procedure 990.08; AOAC, 2005) after nitric acid-perchloric acid wet ash sample preparation (procedure 968.088D; AOAC, 2005). These values were used to calculate ileal digestibility coefficients. Fecal samples were dried in a forced-air oven and finely ground prior to analysis. These samples were analyzed for DM, gross energy, crude protein, Ca, and P.

Data were analyzed as a latin square design using PROC MIXED procedure of SAS (SAS Institute Inc., Cary, N.C.). The model included dietary treatment, animal period and block. Means were separated using pair wise t-tests. Each animal was considered the experimental unit for all calculations. An alpha level of 0.05 was used for determination of statistical significance.

Results

Ileal energy digestibility (FIG. 23A) did not increase with addition of phytase compared with the negative control, but increased by 2.95% and 9.28% with addition of phytase+xylanase+amylase and phytase+xylanase+amylase+lipase, respectively. These changes indicate an effect of 6.3% that was purely due to the addition of lipase on a phytase+xylanase+amylase background. Similarly, ileal digestibility of crude protein did not increase with phytase supplementation compared to the negative control diet (FIG. 23B), but supplementation of phytase+xylanase+amylase significantly increased crude protein digestibility compared to the negative control (+5.6%) and phytase supplementation (+4.0%). Additional supplementation of lipase on top of phytase+xylanase+amylase further increased crude protein digestibility compared to the negative control (+10.7%), phytase supplementation (+9.1%), and phytase+xylanase+amylase (+4.9%).

At the total tract level (FIG. 24), neither inclusion of phytase nor phytase+xylanase+amylase affected energy digestibility. The reduction of the difference from the ileal to the faecal level with inclusion of phytase+xylanase+amylase was not surprising, since microbial activity in the large intestine tends to annul initial differences in energy digestibility in pigs. In contrast, addition of lipase to this combination exhibited a greater faecal energy digestibility coefficient than the other treatments, which suggests the release and absorption of nutrients that otherwise would not be digested even by microorganisms in the large intestine. The combination of phytase+xylanase+amylase+lipase increased total tract energy digestibility by 4.7% compared to the negative control feed.

Relative increments in total Ca (FIG. 25A) and P retention (FIG. 25B) of diets with phytase (+32% and +293%), phytase+xylanase+amylase (+36% and +323%), and phytase+xylanase+amylase+lipase (+45% and 334%, respectively) were also evident in the current study as compared to the negative control diet. Although changes in Ca and P digestibilities with phytase inclusion were expected, additional improvements with xylanase+amylase+lipase addition indicated a major role of lipase in increasing Ca and P retention of diets with phytase, amylase and xylanase. These increments were significantly greater versus the control feed and phytase by itself, which demonstrate an additive effect of the lipase on Ca and P retention.

In conclusion, addition of lipase on top of phytase+amylase+xylanase increased energy and protein digestibility at the ileal level, and energy digestibility at the total tract level compared to the negative control, phytase addition and phytase+amylase+xylanase addition. Similarly, addition of lipase on top of phytase+amylase+xylanase increased total tract Ca and P retention compared to the negative control, and diets with phytase addition. In-vitro examples contained in this patent indicate that these digestibility increments caused by the addition of lipase are mainly due to an increased phytase activity promoted by the competition of lipase for dietary calcium.

Example 9 Free Fatty Acid Production in Corn-Soy Diet at Acidic Conditions

Results from a Study of Free Fatty Acid Production in Corn-Soy Diet by Lipase

Feed samples are incubated at 40° C. with different combinations of lipases and phytases at ˜pH 2.8.

Reagents:

Appropriate reagents and their concentrations were as follows. Phytase assay buffer: 0.25 M Sodium-acetate, 1 mM CaCl₂, 0.01% Tween 20, pH 5.5. Pepsin solution: 0.75 M HCl containing 2000 U/ml pepsin (Sigma P7000). The feed sample used was a basic corn-soy diet containing 60.01% Corn, 31.52% Soybean meal 48, 4% Soy oil, 0.4% Salt, 0.2% DL Methionine, 1.16% Limestone, 1.46% Dicalcium Phosphate, and 1.25% VIT/MIN mix.

Enzymes:

Lipase 3 as described herein. Phyzyme as described herein. TfuIII lipase is a bacterial cutinase from Thermobifida fusca (Tfu_(—)0883; NCBI accession number YP_(—)288944.1) expressed in Bacillus subtilis. TfuIII was analysed using the Lipase assay as described in MATERIALS AND METHODS. Further, the enzyme was analysed using the same assay, though, pH conditions were changed to pH 7.0 instead of pH 5.5. Lipase activity measured at pH 7.0 is described as LIP7U, using the same definition as described in the Lipase assay in MATERIALS AND METHODS. The ratio between lipase activity measured at pH 5.5 (LIPU) and pH 7.0 (LIP7U) was found to be 1:1.4.

Ronozyme P-CT and Natuphos are commercially available fungal phytases. The samples were obtained as dry products. 1 g of the sample was initially resuspended in 50 ml of phytase assay buffer and extracted using a magnetic stirrer for 20 min. The extraction mixture was filled to final 100 ml with phytase assay buffer and filtered through a glass fiber filter. Phytase activity of the final extracts were measured according to the phytase assay described in MATERIALS AND METHODS.

The lipase samples were diluted in H₂O to an enzyme activity of 30 LIPU/ml for Lipase 3 and 30 LIPU/ml for TfuIII.

The phytase samples were diluted in phytase assay buffer to an enzyme activity of 5 FTU/ml.

In Vitro Incubation at Acidic pH:

The following enzyme combinations were evaluated in the in vitro incubation: Lipase 3, Lipase 3+Ronozyme P, Lipase 3+Natuphos, Lipase 3+Phyzyme, TfuIII, TfuIII+Ronozyme P, TfuIII+Natuphos, TfuIII+Phyzyme, and no lipase or phytase.

For each incubation, one g of feed sample was weighed into a 50 ml screw-cap tube. 100 μl of each respective enzyme solution followed by H₂O to 1 ml in total was added to respective tubes. Next, 0.5 ml of Pepsin solution was added to each tube and the content was mixed carefully. Five glass marbles were added before the samples were incubated at 40° C. in a shaking water bath for 45 min. After incubation the tubes were centrifuged for 10 min at 3500 rpm. pH of the supernatant for the “no lipase and no phytase” incubation was measured. Finally, the samples were frozen in liquid nitrogen and placed in the freeze dryer over night. All incubations were performed in duplicate.

Free Fatty Acid Measurement:

The amount of free fatty acid produced during the incubation was measured using a NEFA-HR(2) kit (WAKO Chemicals GmbH). The samples were placed at room temperature and 20 ml of 96% Ethanol was added. The samples were manually resuspended before mixed on a rotary wheel for 1 hour at room temperature. After mixing the samples were centrifuged at 3500 rpm for 10 min. The supernatant was diluted 5 times in 96% Ethanol before free fatty acids in the sample were measured. 110 μl NEFA reagent R1 was equilibrated for 5 minutes at 37° C. before 15 μl diluted sample was added and the reaction mixture was incubated at 37° C. for 10 minutes. 55 μl NEFA reagent R2 was added and incubation continued for another 10 minutes at 37° C. before OD_(520 nm) was measured. The content of free fatty acid was calculated from a standard curve prepared from NEFA Standard (WAKO Chemicals GmbH).

Results:

The results of the free fatty acid measurement are presented in FIG. 29. This Figure shows Free fatty acid production in corn-soy diet. The amount of produced free fatty acids (FFA) is corrected for the blank value without added lipase or phytase. The pH of the incubation with no lipase and no phytase was measured to be pH 2.8 and assumed to be representative of all the incubations in this example. This demonstrates that the incubations were done at very acidic conditions. Lipase 3 alone or in combination with Phyzyme, Natuphos, or Ronozyme P shows a significant release of FFA compared to all the samples with TfuIII, either alone or in combination with Phyzyme, Natuphos, or Ronozyme P. This clearly shows that Lipase 3 would be very effective in fat hydrolysis in the acidic environment in the stomach of a pig or gizzard of a chicken.

Example 10 Effect on Phytase Activity of Adding Calcium to the Assay Mixture and Effect of Adding Free Fatty Acid to an Assay Containing CaCl₂

Results from a study of phytic acid-calcium complex formation with increasing concentration of calcium and regain of the phytase activity by adding free fatty acid.

Background:

Phytic acid can readily form chelates with divalent minerals such as calcium. This phytate-mineral complex may exist as insoluble precipitates or soluble complexes and can potentially be resistant to hydrolysis by phytase. Free fatty acid may potentially function as a competitive chelator, by forming Ca-soaps resulting in decreased formation of phytase-resistant forms of phytic acid and concomitant increase in released phytate-P.

Reagents:

Appropriate reagents and their concentrations were as follows. Assay buffer: 0.25 M Sodium-Acetate, 3% Triton X-100 (w/v), pH 5.5. Phytate solution: 9.1 mM Phytic acid sodium salt hydrate (Sigma P0109) in assay buffer. CaCl₂ solution: 0.3 M CaCl₂ in assay buffer. FFA solution: 40.8 mg/ml Palmitic acid—Stearic acid mixture (Sigma 09586) dissolved in 96% Ethanol. Ethanol: 96% Ethanol.

Enzymes:

Phyzyme as described herein. Ronozyme P-CT and Natuphos are commercially available fungal phytases. BP17 as described herein. The samples were obtained as dry product. 1 g of the sample was initially resuspended in 50 ml of 0.25 M Sodium-acetate, 1 mM CaCl₂, 0.01% Tween 20, pH 5.5 and extracted using a magnetic stirrer for 20 min. The extraction mixture was filled to final 100 ml with 0.25 M Sodium-acetate, 1 mM CaCl₂, 0.01% Tween 20, pH 5.5 and filtered through a glass fiber filter. Phytase activity of the final extracts was measured according to the phytase assay described in MATERIALS AND METHODS.

The phytase samples were diluted in 0.25 M Sodium-acetate, 1 mM CaCl₂, 0.01% Tween 20, pH 5.5 to an enzyme activity of 0.3 FTU/ml.

Phytic Acid Hydrolysis by Phytase:

0.5 ml Ethanol was mixed with 1.4 ml assay buffer and 1 ml phytate solution in a 12 ml test tube. The mixture was equilibrated in a water bath at 37° C. for 5 min before the reaction was started by addition of 0.1 ml phytase solution. For the blank measurement 0.1 ml assay buffer was added instead of phytase solution. After 1 hour incubation at 37° C. the reaction was stopped by addition of 0.75 ml 2.5 M HCl. All incubations were performed in duplicate.

Phytic Acid Hydrolysis by Phytase with Increasing Concentration of CaCl2:

The appropriated volume of CaCl₂ solution, was mixed in a 12 ml test tube with 0.5 ml Ethanol, 1 ml phytate solution and assay buffer to a final assay volume of 3 ml. The mixture was equilibrated in a water bath at 37° C. for 5 min before the reaction was started by addition of 0.1 ml phytase solution. For the blank measurement 0.1 mL assay buffer was added instead of phytase solution. After 1 hour incubation at 37° C. the reaction was stopped by addition of 0.75 ml 2.5 M HCl. All incubations were performed in duplicate.

Phytic Acid Hydrolysis by Phytase in the Presence of 15 mM CaCl₂ and with Increasing Concentration of FFA:

The appropriated volume of FFA solution was mixed in a 12 ml test tube with Ethanol to a volume of 0.5 ml. 0.15 ml Calcium solution, 1 ml phytate solution and assay buffer to a final assay volume of 3 ml was added. The mixture was equilibrated in a water bath at 37° C. for 5 min before the reaction was started by addition of 0.1 ml phytase solution. For the blank measurement 0.1 mL assay buffer was added instead of phytase solution. After 1 hour incubation at 37° C. the reaction was stopped by addition of 0.75 ml 2.5 M HCl. All incubations were performed in duplicate.

Measurement of Phytase Activity:

Inorganic phosphate concentration in the hydrolysis samples, were measured using the Phosphorus reagent (Thermo Scientific) on the Konelab analyzer. 180 μl Phosphorus reagent was mixed with 5 μl sample and incubated for 4 minutes at 37° C. before OD_(340 nm) were measured. The content of phosphate was calculated using the sCal Konelab Calibrator and Nortrol Konelab Control (Thermo Scientific). Phytase activity was calculated as released inorganic phosphate by subtracting the blank measurement from the result. All activities were calculated as relative to the sample with no CaCl₂ present.

Results

FIG. 30 shows the production of a phytase resistant complex of phytic acid and calcium and reversal by adding free fatty acids. FIG. 30A shows the effect on phytase activity of increasing CaCl₂ concentration. FIG. 30B shows the effect on the phytase activity of adding free fatty acids to the reaction mixture containing 15 mM.

The results of the phytic acid hydrolysis with increasing concentration of CaCl₂ are presented in FIG. 30A. Decreasing phytase activity with increasing CaCl₂ concentration is observed for all four phytases. For Phyzyme, BP17, and Ronozyme P reduced activity starts at 5 mM CaCl₂, while Natuphos has increased activity at 5 mM and reduced activity starts at 15 mM CaCl₂. At 20 mM CaCl₂ no phytase activity is observed for Phyzyme, BP17, and Ronozyme P, indicating that at CaCl₂ concentrations above 20 mM all phytic acid is bound in a complex resistant against, at least Phyzyme, BP17, and Ronozyme P hydrolysis. For Natuphos a significant reduction in phytase activity is observed at 30 mM CaCl₂, though a CaCl₂ concentration of 60 mM seems to be necessary to bind all phytic acid in a complex resistant against Natuphos hydrolysis. The results on phytase activity of Phyzyme, BP17, and Ronozyme P of adding free fatty acids to the reaction mixture containing 15 mM CaCl₂ is presented in FIG. 30B. When adding free fatty acid to the reaction mixture containing 15 mM CaCl₂, the phytases regain more and more activity with increasing amount of free fatty acid. Small differences between the three phytases in response to the free fatty acid concentration is observed, though all three regains 100% activity at 25 mM free fatty acid. This clearly demonstrates that free fatty acids can competitively bind calcium, and thereby has the ability to prevent formation of a phytase resistant phytic acid-calcium complex. Free fatty acids may be added as a single component as demonstrated here or can be formed by the action of a lipase on a triglyceride substrate.

Example 11 Lipase Incubation Followed by Phytase Assay—Effect on Phytase Activity of Adding Calcium to the Assay Mixture and Effect of Adding Lipase Reaction Mix to an Assay Containing CaCl2

Results from a study of phytic acid-calcium complex formation with increasing concentration of calcium and regain of the phytase activity by adding free fatty acid produced by Lipase 3.

Background:

In example 11 the effect on phytase activity of adding free fatty acids to a phytase reaction mixture containing 15 mM CaCl₂ was described. In this example a similar experiment is conducted, though the free fatty acids are produced by Lipase 3. A triglyceride emulsion is incubated with Lipase 3, a fraction of this mixture is subsequently added to the phytase reaction mixture containing 30 mM CaCl₂ and the effect on the phytase activity is observed.

Reagents:

Appropriate reagents and their concentrations were as follows. Lipase sample buffer: 50 mM Sodium-acetate, pH 5.0, 0.1% BSA (w/v), 1.2% NaCl (w/v). Triglyceride substrate: 2% Glyceryl Trioctanoate (v/v), 0.3% NaCl (w/v), 13% Triton X-100 (w/v), 120 mM Sodium-Acetate, pH 5.0. Phytase assay buffer: 0.25 M Sodium-Acetate, pH 5.5. Phytate solution: 9.1 mM Phytic acid sodium salt hydrate (Sigma P0109) in assay buffer. CaCl₂ solution: 0.3 M CaCl₂ in assay buffer. Ethanol: 96% Ethanol.

Enzymes:

Lipase 3 and Phyzyme as described herein. The Lipase 3 sample was diluted in Lipase sample buffer to an enzyme activity of 10 LIPU/ml. The phytase sample was diluted in 0.25 M Sodium-acetate, 1 mM CaCl₂, 0.01% Tween 20, pH 5.5 to an enzyme activity of 0.3 FTU/ml.

Lipase Reaction Mixture:

5 ml Triglyceride substrate was mixed with 1.0 ml lipase solution in a 12 ml test tube. For a blank mixture 1.0 ml Lipase sample buffer was added instead of Lipase solution. The mixtures were incubated in a water bath at 30° C. for 120 min with continuous stiffing. The mixtures were directly used as Lipase reaction mixture or Blank reaction mixture, respectively, in the following phytase assay.

Phytic Acid Hydrolysis by Phytase with Increasing Concentration of CaCl2 Including Blank Reaction Mixture:

The appropriate volume of CaCl₂ solution was mixed in a 12 ml test tube with 1.3 ml Blank reaction mixture, 1 ml phytate solution and assay buffer to a final assay volume of 3 ml. The mixture was equilibrated in a water bath at 37° C. for 5 min before the reaction was started by addition of 0.1 ml phytase solution. For the blank measurement 0.1 mL assay buffer was added instead of phytase solution. After 1 hour incubation at 37° C. the reaction was stopped by addition of 0.75 ml 2.5 M HCl.

Phytic Acid Hydrolysis by Phytase in the Presence of 30 mM CaCl2 and Lipase Reaction Mixture:

1.3 ml Blank reaction mixture or Lipase reaction mixture was mixed in a 12 ml test tube with 0.3 ml calcium solution, 1 ml phytate solution and assay buffer to a final assay volume of 3 ml. The mixture was equilibrated in a water bath at 37° C. for 5 min before the reaction was started by addition of 0.1 ml phytase solution. After 1 hour incubation at 37° C. the reaction was stopped by addition of 0.75 ml 2.5 M HCl.

Measurement of Phytase Activity:

Inorganic phosphate concentration in the hydrolysis samples, were measured using the Phosphorus reagent (Thermo Scientific) on the Konelab analyzer. 180 μl Phosphorus reagent was mixed with 5 μl sample and incubated for 4 minutes at 37° C. before OD_(340 nm) were measured. The content of phosphate was calculated using the sCal Konelab Calibrator and Nortrol Konelab Control (Thermo Scientific). Phytase activity was calculated as released inorganic phosphate by subtracting the blank measurement from the result. All activities were calculated as relative to the sample with no CaCl₂ present.

Results

FIG. 31 shows the production of a phytase resistant complex of phytic acid and calcium and reversal by adding Lipase reaction mixture including lipase. FIG. 31A shows the effect on phytase activity of increasing CaCl₂ concentration and FIG. 31B shows the effect on the phytase activity of adding Lipase reaction mixture to the phytase reaction mixture containing 30 mM CaCl₂.

The results of the phytic acid hydrolysis with increasing concentration of CaCl₂ in the presence of a blank lipase reaction mixture (no lipase present) are presented in FIG. 31A. Decreasing phytase activity with increasing CaCl₂ concentration is observed with a significant reduction in phytase activity at 30 mM CaCl₂ and no remaining activity at 40 mM CaCl₂. This calcium inhibition profile is different from the corresponding curve shown in Example 11. The reason is the amount of Triton X-100 applied to emulsify the Triglyceride substrate in the lipase reaction mixture, which results in twice as much Triton X-100 in the phytase reaction mixture as compared to the experiment described in Example 11. The change of Triton X-100 concentration changes the properties for complex formation between the phytic acid and calcium.

The results of adding lipase reaction mixture to the phytase reaction mixture containing 30 mM CaCl₂ is presented in FIG. 31B. Adding the lipase reaction mixture including lipase, results in a significant increase of phytase activity compared to the samples without lipase added.

This clearly demonstrates that free fatty acids produced by lipase activity on a triglyceride substrate can competitively bind calcium, and thereby has the ability to prevent formation of a phytase resistant phytic acid-calcium complex. When the phytic acid-calcium complex is not formed a larger amount of phytic acid is more readily accessible to degradation by the phytase leading to increased phytase activity.

In the animal, this increase in phytase activity as a result of the lipase activity, will lead to increased P and Ca retention as well as increased apparent amino acid digestibility (Ravindran, V. et al, Br. Poult. Sci., 41, 193-200 (2000) and Selle, P. H. et al, Livestock Science, 124, 126-141 (2009)). This will lead to increased P and Ca retention as well as increased energy metabolizability and apparent amino acid digestibility when compared to animals not supplemented the lipase on top of phytase.

Example 12 pH Profile of Lipases in Feed

Results from a Study of the pH Profile of Lipase 3 and Pancreatic Lipase in Feed

Feed samples are incubated at 40° C. with Lipase 3 and pancreatic lipase using different pH buffers to show the pH profile of Lipase 3 and pancreatic lipase.

Enzymes:

Lipase 3 as described herein. Pancreatic lipase as in Pancreatin from porcine pancreas (Sigma P7545). The Lipase 3 sample was diluted in H₂O to an enzyme activity of 300 LIPU/ml. The Pancreatin sample was diluted in H₂O to a concentration of 2 mg/ml.

Reagents:

Appropriate buffers and their concentrations were as follows. 0.25 M HCl; 0.5 M Glycine-HCl, pH 2.5; 0.5 M Glycine-HCl, pH 3.5; 0.5 M Sodium-phosphate, pH 6.5; 0.5 M Sodium-phosphate, pH 7.5; 0.5 M Tris, pH 8.5; 1 M NaHCO₃, pH 10. Other reagents were as follows. TDC solution: Sodium taurodeoxycholate (Sigma T0875) was diluted in H₂O to 80 mM. The feed sample used was a basic corn-soy diet containing 60.01% Corn, 31.52% Soybean meal 48, 4% Soy oil, 0.4% Salt, 0.2% DL Methionine, 1.16% Limestone, 1.46% Dicalcium Phosphate, and 1.25% VIT/MIN mix.

In Vitro Incubation with Different pH Buffers:

For each incubation, one g of corn-soy feed sample was weighed into a 50 ml screw-cap tube. 0.1 ml of enzyme solution followed by 0.1 ml of TDC solution and 1.8 ml of respective buffer was added to respective tubes. Five glass marbles were added before the samples were incubated at 40° C. in a shaking water bath for 120 min. After incubation the tubes were centrifuged for 10 min at 3500 rpm and the pH of the supernatant was measured. Afterwards the samples were frozen in liquid nitrogen and placed in the freeze dryer over night. All incubations were performed in duplicate.

Free Fatty Acid Measurement:

The amount of free fatty acid produced during the incubation was measured using a NEFA-HR(2) kit (WAKO Chemicals GmbH). The samples were placed at room temperature and 20 ml of 96% Ethanol was added. The samples were manually resuspended before mixed on a rotary wheel for 1 hour at room temperature. After mixing the samples were centrifuged at 3500 rpm for 10 min. The supernatant was diluted 5 times in 96% Ethanol before free fatty acids in the sample was measured. 110 μl NEFA reagent R1 was equilibrated for 5 minutes at 37° C. before 15 μl diluted sample was added and the reaction mixture was incubated at 37° C. for 10 minutes. 55 μl NEFA reagent R2 was added and incubation continued for another 10 minutes at 37° C. before OD_(520 nm) was measured. The content of free fatty acid was calculated from a standard curve prepared from NEFA Standard (WAKO Chemicals GmbH).

Results:

FIG. 32 shows the pH profile of Lipase 3 and Pancreatic lipase measured in a corn-soy diet. The concentration of Lipase 3 was 30 LIPU/g of feed and the concentration of Pancreatic lipase was 0.2 mg/ml of feed. The activity is shown as relative to the highest measured amount of produced free fatty acid.

The pH in the different buffers was as follows for Lipase 3 and pancreatic lipase, respectively: 0.25 M HCl provides pH 3.41 and pH 3.02; 0.5 M Glycine-HCl, pH 2.5 provides pH 3.99 and pH 3.76; 0.5 M Glycine-HCl, pH 3.5 provides pH 4.93 and pH 5.18; 0.5 M Sodium-phosphate, pH 6.5 provides pH 6.45 and pH 6.45; 0.5 M Sodium-phosphate, pH 7.5 provides pH 7.26 and pH 7.22; 0.5 M Tris, pH 8.5 provides pH 8.33 and pH 8.18; 1 M NaHCO₃, pH 10 provides pH 9.95 and pH 10.07. For Lipase 3 the pH optimum in feed is found at pH 4.0 with decreasing activity at both lower and higher pH. pH 3.4 is the lowest pH evaluated and the activity will probably continue to decrease with further decreasing pH. For the pancreatic lipase the pH optimum in feed is found at pH 8.2 with decreasing activity at both lower and higher pH. The conclusion is that Lipase 3 has a pH profile very different from the pH profile of the pancreatic lipase. Further, the pH profile of Lipase 3 indicates that Lipase 3 could be very effective in fat hydrolysis in the acidic environment in the stomach of a pig or gizzard of a chicken.

Example 13 Ileal Digestibility and Total Tract Retention of Phosphorus and Calcium of Diets Supplemented with Lipase, Phytase and the Combination of Lipase+Phytase Versus a Control Diet in Broiler Chickens

Materials and Methods

Experiments were conducted to evaluate the ileal digestibility, and the total tract retention of P and Ca in broiler chickens fed diets supplemented with lipase or phytase by themselves, and lipase and phytase in combination, versus a negative control diet. Diets (Table 4) were based on corn and soybean meal, and contained corn DDGS, corn gluten meal, and soybean oil.

TABLE 4 Composition and calculated analysis (g/kg as fed) of the diets Pre-starter Negative control Ingredient Corn 498.3 496.8 Corn DDGS 50.0 50.0 Corn gluten meal, 60% 50.0 50.0 Soybean meal, 48% 311.9 312.2 Maize/Wheat starch 2.0 2.0 Soybean oil 40.0 40.0 Lysine HCl 3.8 3.8 DL-methionine 2.7 2.7 L-threonine 1.0 1.0 Inert marker 3.0 3.0 Salt 3.6 3.6 Limestone 13.8 3.0 Dicalcium phosphate 16.4 13.5 Trace mineral- vitamin premix 3.5 3.5 Space (Enzyme) 0.0 14.9 Calculated analysis Metabolisable energy, kcal/kg 3.140 3.100 Crude protein, g/kg 240 240 Digestible lysine, g/kg 12.6 12.6 Digestible methionine, g/kg 6.4 6.4 Digestible methionine + cysteine, g/kg 9.0 9.0 Digestible threonine, g/kg 7.9 7.9 Calcium, g/kg 10.5 5.7 Available phosphorus, g/kg 4.3 3.8

The experimental design consisted of 6 dietary treatments, each randomly allocated to 6 replicate cages (8 birds per cage) as follows:

1. Negative control (NC)

2. NC+lipase (1,000 LIPU/kg)

3. NC+lipase (3,000 LIPU/kg)

4. NC+phytase (500 FTU/kg)

5. NC+phytase (500 FTU/kg)+lipase (1,000 LIPU/kg)

6. NC+phytase (500 FTU/kg)+lipase (3,000 LIPU/kg)

Day-old broiler chicks were randomly assigned to electrically heated battery brooders in an environmentally controlled room. The chicks received constant fluorescent illumination and were allowed free access to the diets and water. The birds received a pre-starter diet until day 14. At this age, birds were assigned to dietary treatments on the basis of body weight, and transferred to grower cages. Birds were fed the experimental diets from day 14 until day 21. The temperature was maintained at 31° C. for the first week and then gradually reduced to 22° C. by the third week.

Feed intake and total excreta output of each cage were measured from day 17 to 20 post-hatch. Excreta from each cage were pooled, mixed in a blender and sub-sampled. Each sub-sample was freeze-dried, ground to pass through a 0.5 mm sieve and stored in airtight plastic containers at −4° C. pending analysis. The diets and excreta samples were analysed for dry matter (DM), calcium (Ca), and phosphorous (P).

On d 21, two birds from each replicate cage were selected and euthanised by intra-cardial injection of sodium pentabarbitone. The small intestine immediately was exposed and the contents of the lower half of the ileum were collected by gently flushing with distilled water into plastic containers. The ileum was defined as that portion of the small intestine extending from vitelline diverticulum to a point 40 mm proximal to the ileo-caecal junction. Digesta were pooled within a cage and were lyophilised, ground to pass through a 0.5-mm sieve, and stored at −4° C. in airtight containers until laboratory analysis for DM, titanium, Ca, and P.

Data were analyzed by ANOVA using PROC MIXED procedure of SAS (SAS Institute Inc., Cary, N.C.). The model included the fixed effect of dietary treatment and the random effect of block. Means were separated using pair wise t-tests. Each pen was considered the experimental unit for all calculations. An alpha level of 0.05 was used for determination of statistical significance.

Results

FIG. 33 shows that phytase did not significantly increase phosphorus ileal digestibility compared to the negative control, but produced a numeric increment of 6.0% as it was expected. Lipase, both at 1,000 and 3,000 LIPU/kg, produced no effect on ileal phosphorus digestibility compared to the negative control. In contrast, the combination of lipase+phytase was able to significantly increase the ileal phosphorus digestibility compared to the negative control by 14.3% at 1,000 LIPU/kg and 10.6% at 3,000 U/kg of lipase inclusion. The combination of lipase+phytase significantly increase ileal phosphorus digestibility compared to the phytase treatment, only when lipase was included at 1,000 u/kg by a difference of 7.8%.

FIG. 34 shows that there are no significant effects on ileal calcium digestability of any of the enzyme treatments compared to the negative control diet. Nonetheless, the numerically highest Ca digestibility values were obtained with the combinations phytase+lipase, which exhibited values that were 118.3% and 115.6% of the values obtained with the negative control diet, when lipase was included at 1,000 u/kg and 3,000 u/kg, respectively.

FIG. 35 shows that neither lipase inclusion nor phytase inclusion resulted in any significant differences in total tract phosphorus retention compared to the negative control. Nonetheless, phytase produced a numeric increment of phosphorus retention of 9.7% compared to the negative control. The combination of lipase+phytase significantly increased phosphorus retention compared to the negative control by 13.4% and 16.0% when lipase was supplemented at 1,000 and 3,000 LIPU/kg, respectively.

FIG. 36 shows that neither lipase inclusion nor phytase inclusion results in any significant differences in total tract calcium retention compared to the negative control. However, the combination of phytase and 3,000 LIPU/kg significantly increased the Ca retention by 17.7% compared with the negative control.

In synthesis, neither lipase nor phytase produced significant effects of P and Ca digestibility in the current study. However, the combination of phytase and 1,000 LIPU/kg of lipase increased ileal and total tract phosphorus retention compared to the negative control, and ileal phosphorus digestibility compared to phytase inclusion. The combination of phytase and 3,000 LIPU/kg of lipase increased ileal and total tract phosphorus retention, and total tract Ca retention compared to the negative control. These incremental effects of lipase on top of phytase in absorption and utilization of calcium and phosphorus appear to be mainly related to the production of free fatty acids by lipase in the gastro intestinal tract, which promotes competition for calcium and improves phytase activity, as confirmed in in-vitro examples described herein. 

The invention claimed is:
 1. A method of making a feed supplement comprising mixing a lipolytic enzyme and a phytase, the feed supplement used to increase an uptake of at least one nutrient in a feedstuff to which the feed supplement is added, wherein said lipolytic enzyme has a lipase activity at a pH in a range from pH 1.5 to pH 3.5.
 2. The method of claim 1, further comprising the additional step of homogenising the feed supplement.
 3. The method according to claim 1, further comprising the additional step of pelleting the feed supplement.
 4. The method of claim 1, wherein said lipolytic enzyme comprises one or more of: a polypeptide having sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a polypeptide having at least 70% identity to SEQ ID NO: 7 or SEQ ID NO: 8; a polypeptide produced by expression of a nucleotide sequence comprising a sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and a sequence having at least 70% identity to SEQ ID NO: 9 or SEQ ID NO:
 10. 5. The method of claim 4, wherein the lipolytic enzyme is produced by expressing a nucleotide sequence comprising a sequece of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having at least 70% identity to SEQ ID NO: 9 or SEQ ID NO: 10 in a Trichoderma reesei host cell.
 6. The method of claim 1, wherein the phytase comprises one or more of an E. coli phytase having a sequence of SEQ ID NO: 13 and a Buttiauxella phytase having a sequence of one of SEQ ID NOs 1-6, or a polypeptide having at least 70% identity to one of SEQ ID NOs 1-6, or a polypeptide produced by expression of a nucleotide sequence comprising a sequence of SEQ ID NO: 11, or a sequence comprising nucleotides 253 to 1483 of SEQ ID NO:14, or a sequence that has at least 70% identity to SEQ ID NO: 11 or nucleotides 253 to 1483 of SEQ ID NO:14.
 7. The method of claim 1, wherein said phytase has phytase activity at a pH in a range from pH 2.5 to pH 5.5.
 8. A method of making a feedstuff comprising adding to a feed material a feed supplement that comprises at least one phytase and at least one lipase having a lipase activity at a pH in a range from pH 1.5 to pH 3.5, the feedstuff for increasing an availability of at least one nutrient, increasing an available metabolic energy from the feed material, or increasing the availability of the at least one nutrient and increasing the available metabolic energy from the feed material.
 9. The method of claim 8, further comprising adding at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, anti-foam, Na₂SO₄, Talc, PVA and mixtures thereof.
 10. The method of claim 8, further comprising homogenising the feed supplement.
 11. The method of claim 8, further comprising pelleting the feed supplement.
 12. The method of claim 8, wherein the feedstuff further comprises at least one low fibre feed material, wherein the at least one low fibre feed material is one of corn, wheat, an animal by-product meal, or soybean.
 13. The method of claim 8, wherein the at least one nutrient is selected from one or more of phosphorous, calcium, amino acids, fat, and starch. 